Protocol for antibody optimization and panel design in high-dimensional multiplexed immunofluorescence imaging

Summary

The rise of spatial biology technologies is advancing our ability to study cellular interactions within native tissue contexts using antibody-based multiplexed imaging. Here, we present a protocol for designing and optimizing a suitable antibody panel, with a specific focus on the PhenoCycler-Fusion system. We outline steps for antibody selection, optimization, and validation, as well as reporter plate preparation and cycle generation for image acquisition. This protocol incorporates key considerations for robust panel development for spatial profiling of tissues.

For complete details on the use and execution of this protocol, please refer to Abraham et al.¹

Graphical abstract

Highlights

• •Steps for selecting antibodies for immunofluorescence imaging of tissues
• •Guidance for validating antibody performance in single and multiplexed imaging formats
• •Recommendations for assigning barcodes based on autofluorescence and antigen abundance
• •Steps for reporter plate setup and cycle design for imaging with PhenoCycler-Fusion

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

The rise of spatial biology technologies is advancing our ability to study cellular interactions within native tissue contexts using antibody-based multiplexed imaging. Here, we present a protocol for designing and optimizing a suitable antibody panel, with a specific focus on the PhenoCycler-Fusion system. We outline steps for antibody selection, optimization, and validation, as well as reporter plate preparation and cycle generation for image acquisition. This protocol incorporates key considerations for robust panel development for spatial profiling of tissues.

Before you begin

The rapid advancements in spatial biology techniques have enabled researchers to interrogate cell-cell interactions and molecular organization within intact tissue architecture, providing key insights into the complexity of health and disease.^1,2,3,4 Among these technologies, antibody and DNA-based multiplexed imaging platforms such as the PhenoCycler-Fusion (PCF) currently allow simultaneous visualization of up to 101 markers,⁵ on a single tissue section at sub-cellular resolution. This platform functions by conjugating antibodies to unique oligonucleotide barcodes for use in staining tissues.⁶ The PCF instrument then automates iterative cycles of tissue washing, hybridization with fluorescently labeled complementary oligonucleotides, imaging, and stripping to generate highly multiplexed imaging datasets.⁶

Although the commercial provider for PCF, Akoya Biosciences, offers pre-validated antibody panels, many research groups require customized panels tailored to specific biological questions or tissue types. These pre-validated antibody panels are composed of pre-conjugated antibodies, restricting them to the PCF workflow and making them unsuitable for use in other imaging contexts. This lack of flexibility underscores the importance of standardized and reproducible workflows for antibody selection, optimization, and validation on the tissue of interest.

The protocol below details steps for designing and optimizing a custom antibody panel for multiplexed imaging using the PCF. These steps were developed using formalin-fixed paraffin embedded (FFPE) tissues, and while they can be adapted to fresh-frozen tissues or other sample types, additional optimization is required to account for tissue-dependent differences in antigen accessibility and background signal. Moreover, this antibody optimization pipeline can be applied to alternative platforms that use conjugated antibodies (e.g., Imaging Mass Cytometry (IMC)) or experimental contexts that rely on single or multiplexed immunofluorescence imaging of tissues.

Innovation

This protocol provides a comprehensive workflow for the design and optimization of custom antibody panels for multiplexed imaging, with broad applicability beyond a single platform. While the protocol focuses on the PCF system, the strategies outlined for antibody screening, validation, and fluorophore selection are relevant to various immunofluorescence-based approaches, including single-plex, conventional multiplex (2-4 markers), and highly multiplexed spatial proteomics. The implementation of a standardized pipeline will allow researchers to streamline panel development, whilst maintaining flexibility to include markers relevant to their unique experimental context.

These optimization steps are compatible with a range of sample types and imaging platforms, including IMC, as demonstrated in our previous work on human breast cancer.⁷ However, this pipeline is specifically optimized for the PCF system, and certain steps, such as DNA-barcode conjugation, are unique to this platform. Alternative multiplexing platforms require platform-specific adjustments. For example, platforms that use directly fluorophore- or metal-tagged antibodies do not require barcode conjugation and instead place greater emphasis on channel separation, staining order, or signal amplification rather than on barcode assignment and fluorophore compatibility.

Importantly, while existing protocols detail how to perform staining and imaging using systems such as the PCF, they often begin with the assumption that users have access to fully validated antibody panels, which is not generally the case. This protocol addresses an unmet need by describing the foundational steps involved in developing such panels. By standardizing the upstream panel development process, this protocol enables its users to generate high-quality, reproducible data tailored to their unique experimental questions. For example, this work builds upon the study by Abraham et al.¹, by providing a systematic optimization workflow necessary to generate the type of spatial data analyses presented in that publication.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Anti-alpha smooth muscle actin antibody (clone 1A4)	Abcam	Cat# ab203696; RRID: AB_262054
Anti-CD19 antibody (clone EPR23174145)	Abcam	Cat# ab245235; RRID: AB_2895109
Anti-PD-L1 antibody (clone D5V3B)	Cell Signaling Technologies	Cat# 64988; RRID: AB_2799672
AF546 goat anti-rabbit IgG	Invitrogen	Cat# A-11035; RRID: AB_2534093
AF555 donkey anti-mouse IgG	Invitrogen	Cat# A-31570; RRID: AB_2536180
AF647 donkey anti-rabbit IgG	Invitrogen	Cat# A-31573; RRID: AB_2536183
AF647 goat anti-mouse IgG	Invitrogen	Cat# A-21235; RRID: AB_2535804
AF750 goat anti-rabbit IgG	Invitrogen	Cat# A-21039; RRID: AB_2535710
AF750 goat anti-mouse IgG	Invitrogen	Cat# A-21037; RRID: AB_2535708
Chemicals, peptides, and recombinant proteins
Xylenes	Sigma-Aldrich	Cat# 534056
Ethanol	Greenfield Global	Cat# P016EAAN
Methanol	Greenfield Global	Cat# 339WORLDCS4L
Tris	Bio Basic	Cat# TB0195
EDTA	Bio Basic	Cat# EB0185
Polysorbate/Tween 20	VWR Chemicals	Cat# 0777-1L
Sodium chloride (NaCl)	Bio Basic	Cat# SB0476
Potassium chloride (KCl)	Bio Basic	Cat# PB0440
Sodium hydroxide (NaOH) 10 N	VWR Chemicals	Cat# BDH7247-1
50% H2O2	Sigma-Aldrich	Cat# 516813
16% paraformaldehyde (PFA)	Electron Microscopy Sciences	Cat# 15710
Normal donkey serum	Jackson ImmunoResearch	Cat# 017-000-121
1X D-PBS	Wisent	Cat# 311-425-CL
DAPI (1 mg/mL)	Thermo Scientific	Cat# 62248
Fluoromount-G	Invitrogen	Cat# 00-4958-02
ProLong	Thermo Scientific	Cat# P36961
Dimethyl sulfoxide (DMSO)	Fisher Scientific	Cat# BP231-1
Laemmli loading dye	Bio-Rad	Cat#1610737EDU
GelCode blue stain reagent	Thermo Scientific	Cat# 24590
Salmon sperm DNA	Fisher Scientific	Cat# AM9680
Critical commercial assays
Conjugation kit (contains reagents for antibody conjugation, including filter blocking solution, reduction solutions 1 & 2, conjugation solution, purification solution, and antibody storage solution)	Akoya Biosciences	Cat# 7000009
Staining kit (includes reagents for tissue staining, such as hydration buffer, staining buffer, storage buffer, blockers N/J/G/S, and fixative reagent)	Akoya Biosciences	Cat# 7000008
10X buffer kit (contains the 10X PhenoCycler buffer and buffer additive)	Akoya Biosciences	Cat# 7000019
Black-walled 96-well plate	Akoya Biosciences	Cat# 7000006
96-well plate seal	Akoya Biosciences	Cat# 7000007
Flow cell	Akoya Biosciences	Cat# 240204
Barcodes	Akoya Biosciences	Variable
Reporters	Akoya Biosciences	Variable
Other
Hydrophobic barrier PAP pen	Thermo Scientific	Cat# R3777
SuperFrost Plus slides	Fisher Scientific	Cat# 22-037-246
24 × 55 mm no. 1.5 thickness cover Slips	Epredia	Cat# 152455
Kimwipes	Fisher Scientific	Cat# 06-666-1A
1.5 mL Snaplock microcentrifuge tube, amber	Fisher Scientific	Cat# 14-222-164
50 kDa MWCO centrifugal filter units	Fisher Scientific	Cat# UFC505096
0.5 mL screw-cap microtubes	Progene	Cat# 71-050-SKCS
Microtome	Leica	Cat# RM2125 RTS
PT link for pre-treatment	Agilent
LED lamps		20,000 lux intensity
ChemiDoc	Bio-Rad
AxioScan 7	Zeiss
PhenoCycler-Fusion	Akoya Biosciences
QuPath	Bankhead et al.8

RRID: Research Resource Identifiers.

Materials and equipment

10X Tris-EDTA antigen retrieval buffer, pH 9.0

• •To prepare 10X Tris-EDTA antigen retrieval buffer, weigh out 6.05 g Tris and 1.85 g EDTA.
• •Add Tris and EDTA to 400 mL of ddH₂O. Mix with a magnetic stirrer.
• •Adjust pH to 9.0 and complete to 500 mL with ddH₂O.

Reagent	Final concentration	Amount
Tris	100 mM	6.05 g
EDTA	10 mM	1.85 g
ddH2O	N/A	To 500 mL
Total	N/A	500 mL

Store at 4°C for up to 30 days.

1X Tris-EDTA antigen retrieval buffer, pH 9.0

• •Prepare 1X antigen retrieval buffer by adding 150 mL of 10X solution to 1350 mL of ddH₂O.
• •Add 750 μL of Tween 20 and mix well.

Reagent	Final concentration	Amount
10X Tris-EDTA Buffer pH 9.0	N/A	150 mL
ddH2O	N/A	1350 mL
Tween 20	N/A	750 μL
Total	N/A	1500 mL

Mix well and make fresh.

CRITICAL: Tween 20 is viscous. Pipette it slowly using the “reverse pipetting method”.

Alternatives: Tween 20 can be substituted by Triton-X-100.

Alternatives: A pH 6.0 Citrate buffer can be used instead of Tris-EDTA Buffer for antigen retrieval. To prepare 10X Citrate buffer, dissolve 14.7 g of Sodium citrate in 400 mL of ddH₂O, adjust the pH to 6.0, and complete to 500 mL with ddH₂O. The 1X working solution can be prepared in the same way as for 1X Tris-EDTA buffer.

10X Tris-buffered saline

• •Prepare TBS by weighing 80 g of NaCl, 2 g of KCl, and 30 g of Tris.
• •Subsequently add the weighed reagents to 900 mL of ddH₂O. Mix with a magnetic stirrer.
• •Adjust pH to 7.4 and complete to 1000 mL with ddH₂O.

Reagent	Final concentration	Amount
NaCl	1.37 M	80 g
KCl	26.83 mM	2 g
Tris	0.248 M	30 g
ddH2O	N/A	To 1000 mL
Total	N/A	1000 mL

IF wash buffer

• •Prepare the wash buffer by adding 200 mL of 10X TBS to 800 mL of ddH₂O.
• •Add 250 μL of Tween 20 and mix well.

Reagent	Final concentration	Amount
10X TBS	20%	200 mL
ddH2O	N/A	800 mL
Tween 20	N/A	250 μL
Total	N/A	1000 mL

Store at 22°C–25°C for up to 3 days.

CRITICAL: Tween 20 is viscous. Pipette it slowly using the “reverse pipetting method”.

Alternatives: Tween 20 can be substituted by Triton-X-100.

Bleaching solution

• •Prepare the bleaching solution by adding 0.8 mL of 10 N NaOH and 2.7 mL of 50% H₂O₂ in 26.5 mL of PBS.
• •Vortex to mix well.

Reagent	Final concentration	Amount
10 N NaOH	0.27 N	0.8 mL
50% H2O2	4.5%	2.7 mL
1X PBS	N/A	26.5 mL
Total	N/A

Prepare fresh (H₂O₂ is not stable).

CRITICAL: H₂O₂ is irritant, corrosive, and oxidizing. Wear appropriate personal protective equipment and ensure that H₂O₂ is not released into the environment.

Primary blocking buffer

• •Prepare the primary blocking solution by adding 20 μL of 100% (normal) donkey serum to 1000 μL of IF wash buffer.
• •Vortex to mix well.

Reagent	Final concentration	Amount
Normal Donkey Serum	2%	20 μL
IF Wash Buffer	N/A	To 1000 μL
Total	N/A	1000 μL

Mix well and prepare fresh for each experiment.

Antibody dilution buffer

• •Prepare the antibody dilution buffer by adding 1 μL of 100% (normal) donkey serum to 1000 μL of IF wash buffer.
• •Vortex to mix well.

Reagent	Final concentration	Amount
Normal Donkey Serum	0.1%	1 μL
IF Wash Buffer	N/A	To 1000 μL
Total	N/A	1000 μL

Mix well and prepare fresh for each experiment.

Prepared DAPI

• •Prepare DAPI by adding 1 μL of 1 mg/mL DAPI to 500 μL of PBS.
• •Vortex to mix well.

Reagent	Final concentration	Amount
DAPI (1 mg/mL)	2 μg/mL	1 μL
1X PBS	N/A	To 500 μL
Total	N/A	500 μL

Mix well and prepare fresh for each experiment.

CRITICAL: Keep protected from light following preparation.

Antibody reduction master mix

• •Prepare the reduction mix (for 1 conjugation) using reagents from the Conjugation Kit provided from Akoya Biosciences.
• •Add 6.6 μL and 275 μL of Reduction Solution 1 and 2, respectively.
• •Vortex to mix well.

Reagent	Final concentration	Amount
Reduction Solution 1	N/A	6.6 μL
Reduction Solution 2	N/A	275 μL
Total	N/A	281.6 μL

CRITICAL: Do not re-use thawed aliquots of Reduction Solution 1.

Staining buffer with blockers

• •Prepare the antibody dilution buffer (for 2 samples) using reagents from the Staining Kit provided from Akoya Biosciences.
• •Add 9.5 μL of each blocker (N, G, J, and S) to 362 μL of Staining buffer.
• •Vortex to mix well.

Reagent	Final concentration	Amount
N Blocker	Proprietary	9.5 μL
G Blocker	Proprietary	9.5 μL
J Blocker	Proprietary	9.5 μL
S Blocker	Proprietary	9.5 μL
Staining Buffer	N/A	362 μL
Total	N/A	400 μL

Note: The exact composition of the commercially available blockers is proprietary. However, Black et al.⁶ have published a protocol for staining and performing the CODEX (now PhenoCycler) multicycle imaging procedures, including details on the blocking strategies they employed.

1X PhenoCycler Buffer

Reagent	Final concentration	Amount
10X PhenoCycler Buffer	10%	25 mL
ddH2O	90%	225 mL
Total	N/A	250 mL

Hybridization buffer

• •Prepare the hybridization buffer by adding 20 mL of DMSO to 80 mL of 1X PhenoCycler buffer.
• •Mix well.

Reagent	Final concentration	Amount
DMSO	20%	20 mL
1X PhenoCycler Buffer	80%	80 mL
Total	N/A	100 mL

Prepare fresh for each experiment but allow to equilibrate to room temperature (∼22°C–25°C) before use.

Stripping buffer

• •Prepare the stripping buffer by adding 75 mL DMSO to 25 mL of 1X PhenoCycler buffer.
• •Mix well.

Reagent	Final concentration	Amount
DMSO	75%	75 mL
1X PhenoCycler Buffer	25%	25 mL
Total	N/A	100 mL

Prepare fresh for each experiment but allow to equilibrate to room temperature (∼22°C–25°C) before use.

Reporter stock solution

• •Prepare the reporter stock solution (for 5 cycles) using reagents from Akoya Biosciences.
• •Add 5 μL of 1 mg/mL DAPI, 125 μL of salmon sperm DNA, to 150 μL of 10X PhenoCycler buffer and Buffer additive diluted in 1120 μL of nuclease free water.
• •Vortex to mix well.

Reagent	Final concentration	Amount
1 mg/mL DAPI	3.33 μg/mL	5 μL
10 mg/mL Salmon Sperm DNA	0.833 mg/mL	125 μL
10X PhenoCycler Buffer	1X	150 μL
Buffer Additive for PhenoCycler-Fusion	1X	150 μL
Nuclease Free Water	N/A	1070 μL
Total	N/A	1500 μL

Post-staining fixing solution

• •Prepare the post-staining fixative solution (for 2 samples) using reagents from the Staining Kit provided from Akoya Biosciences.
• •Add 1 mL of 16% PFA to 9 mL of Storage buffer.
• •Vortex quickly to mix well.

Reagent	Final concentration	Amount
16% PFA	1.6%	1 mL
Storage Buffer	N/A	9 mL
Total	N/A	10 mL

Final fixative solution

• •Prepare the final fixative solution using reagents from the Staining Kit provided from Akoya Biosciences.
• •Add 20 μL of fixative reagent to 1000 μL of 1X PBS.
• •Vortex quickly to mix well.

Reagent	Final concentration	Amount
Fixative Reagent	Proprietary	20 μL
1X PBS	N/A	1000 μL
Total	N/A	1020 μL

CRITICAL: Do not re-use thawed aliquots of Fixative Reagent.

Step-by-step method details

Experimental design

Timing: Variable

When planning a multiplexed antibody-based imaging experiment, begin by clearly defining the scientific question (Figure 1). This question will guide all subsequent steps, including antibody selection, testing, panel design, and image acquisition parameters.

• 1.
  Define the scientific question to be addressed using multiplexed antibody-based imaging.

  • a.
    Identify the tissue, disease model, or condition under study.
  • b.
    Specify the biological process, cellular interaction, or structure to be investigated.
  • c.
    Define experimental details and the expected outcome of the study (i.e., specify in advance what information you expect to obtain or what hypothesis you aim to test).

    Note: This could include, for example, determining whether immune cell infiltration patterns link to a disease mechanism, identifying a candidate biomarker, or evaluating a therapeutic target.

  • d.
    Ensure alignment with downstream analysis methods, verifying that the type of spatial data and computational outputs will be able to address the question.
• 2.
  Determine experimental constraints, including:

  • a.
    Budget.
  • b.
    Number and type of samples, including biological replicates.
  • c.
    Whether multiplexed imaging is the primary focus or part of a broader workflow.
  • d.
    Available technical support and equipment.

Note: Consider factors such as tissue area, sample format, desired resolution, data analysis pipeline, number of markers, reagent availability, and tissue autofluorescence, as these will govern the optimization required. For example, tissues with high autofluorescence (e.g., liver) may require additional optimization compared to those with low-autofluorescence (e.g., tonsil).

• 3.Revise the scientific question, as needed, based on these constraints.
• 4.Create a list of the antibodies and markers required to address the scientific question by considering downstream analytical requirements.

Note: The number and type of antibodies will vary depending on the biological question and practical constraints.

Note: Ranking and categorizing these markers as essential or desirable can streamline panel development and reduce overall costs.

Figure 1.

Workflow for antibody-based imaging panel design

Flowchart illustrating steps that are broadly applicable across antibody-based imaging platforms (shown in sage green on the left) compared to those specific to the PhenoCycler workflow (shown in light green on the right). General steps include experimental design, antibody selection, and validation principles, while PhenoCycler-specific steps include barcode assignment, antibody conjugation, and reporter cycle design. This schematic highlights the integration of platform-independent considerations with system-specific requirements.

Antibody selection

Timing: Variable

The choice of antibodies is a critical step in building a multiplexed imaging panel. Selection criteria should align with the imaging platform being employed. For systems that require antibody conjugation (e.g., PCF), carrier-free, purified antibodies are necessary.

• 5.
  Search for the desired antibody:

  • a.
    Check if the chosen antibody is available through commercial sources (e.g., Akoya Biosciences antibody database https://www.akoyabio.com/phenocycler/assays/antibodies/) or previously validated for multiplexed imaging (e.g., IBEX protocol https://ibeximagingcommunity.github.io/ibex_imaging_knowledge_base/reagent_resources.html).
  • b.
    If targeting human antigens, users can, when feasible, consult institutional pathology cores to identify clinically validated clones commonly used in diagnostic pathology.

Note: Considering how each antibody will integrate into the panel, including cross-reactivity, fluorophore brightness, and target abundance is important as these factors can impact downstream panel performance and multiplexed imaging quality.

• 6.
  Consult primary literature to identify validated antibodies relevant to your target and application. Some metrics to consider include:

  • a.
    The number of independent publications reporting use of the antibody, whereby a higher number correlates with greater confidence.
  • b.
    The reported signal-to-noise ratio (SNR) or contrast between positive and negative controls.
  • c.
    Specificity and background staining, whereby there is low off-target binding and minimal nonspecific signal in the negative controls.

Note: When reviewing the literature, prioritize antibodies that include appropriate positive and negative controls.

• 7.Use antibody search engines (see Table 1) to compare options across vendors.
• 8.
  Assess antibody compatibility based on the following factors:

  • a.
    Preservatives and formulation:

    • i.
      Avoid antibodies with a high glycerol content or presence of BSA when conjugation for PCF is required. It is best to only select and test antibodies with an available carrier-free option. If unavoidable, contact the vendor for a carrier-free version or take additional steps to perform affinity purification before conjugation (see potential solution 3). If antibodies are not being conjugated (i.e., in standard IF for testing), antibody formulation is a less important consideration.

      CRITICAL: BSA competes with antibodies during oligonucleotide conjugation and reduces conjugation efficiency.

      Note: If pre-purified antibodies are not commercially available, a purification process must be performed prior to conjugation (see potential solution 3).

  • b.
    Clonality:

    • i.
      Monoclonal antibodies are generally preferred due to their specificity and reproducibility.⁹ Polyclonal antibodies may be better used for detecting protein isoforms or post-translational modifications but can introduce batch variability and potentially nonspecific binding.

      Note: When using polyclonal antibodies, request a single-lot batch from the vendor to minimize variability.

  • c.
    Isotype:

    • i.
      IgG antibodies are generally preferable to IgM antibodies, when available, due to better performance in most conjugation and imaging protocols.
  • d.
    Validation status:

    • i.
      Confirm the antibody has been validated for the species and application of interest. Incomplete validation data can be indicative of low-quality reagents or lack of vendor support.

Table 1.

Comparison of antibody search platforms

Search engine	# Of antibodies	Citations listed	Notable features	Link
Antibodies.com	>78,000	Yes	Identifies identical antibodies across vendors; lists lowest price	https://www.antibodies.com
Antibodies-online	>676,000	Yes	Provides independent antibody validation data	https://www.antibodies-online.com
Antibodypedia	>3 million	Yes	Uses a scoring system based on supplier and independent validation	https://www.antibodypedia.com
Antibody Registry	>3 million	Yes	Supports RRID system; includes antibodies from academic laboratories	https://www.antibodyregistry.org
BenchScia	>4.6 million	Yes	Filters published data by experimental contexts	https://www.benchsci.com
Biocompare	>4 million	Yes	Aggregates antibody listings from multiple commercial suppliers	https://www.biocompare.com
Bioza	Not listed	Yes	Independently ranks antibodies using a citation-based rating system	https://www.bioz.com
BIOZOL	>6 million	No	Offers centralized access to a wide range of suppliers	https://www.biozol.de
CiteAba	>8 million	Yes	Ranks antibodies by number of supporting citations in published literature	https://www.citeab.com
IHC World	>18,000	No	Focuses on antibodies validated for IHC	https://ihcworld.com
Labome	>400,000	Yes	Highlights antibodies validated in knockout studies	https://www.labome.com
YCharOS	>850	Yes	Open science platform characterizing commercial antibodies using knockout cell lines and standardized WB/IP/IF assays	https://ycharos.com

^aRequires an account to access information.

Antibody testing

Timing: 7.5–23.5 h

Here, we describe the protocol for performing an immunofluorescence staining on FFPE tissues to test and optimize the selected antibody.

• 9.
  Perform deparaffinization and antigen retrieval.

  Note: This step utilizes xylene to solubilize and remove the paraffin used to embed the tissue, followed by removal of xylene with ethanol. The sample is then rehydrated through increasingly dilute concentrations of ethanol, ending with a final rinse in water. Heat-induced epitope retrieval is performed in basic pH buffer, to remove the methylene bridges formed between proteins by formaldehyde that prevent epitope recognition by primary antibodies.

  • a.
    Following FFPE preservation, mount 4 μm microtome tissue sections onto SuperFrost Plus slides (Fisherbrand).

    Alternatives: Other slide options, such as Leica’s Apex Superior Adhesive slides, may be used; however, the intended application should be carefully considered before purchase. Positively charged slides are generally recommended to ensure an ideal surface for sample adhesion. Additionally, it is important to consult the guidelines of the downstream multiplexed platform being used. For instance, the PhenoCycler requires the use of positively charged glass slides for FF and FFPE tissue section preparation.

  • b.
    Chemical safety hood preparation.

    • i
      In a chemical safety hood, prepare 8 slide staining jars with racks containing approximately 200 mL of xylene (3 jars), 100% ethanol (2 jars), 95% ethanol, 70% ethanol, and 50% ethanol.
    • ii
      Place the slides in a rack with a handle.
  • c.
    Perform the following incubations to deparaffinize the slides, by transferring the slide rack from one staining jar to the next, ensuring the slides are submerged in the solutions:

    • i
      Xylene for 5 min.
    • ii
      Xylene for 5 min.
    • iii
      Xylene for 5 min.
    • iv
      100% ethanol for 5 min.
    • v
      100% ethanol for 5 min.
    • vi
      95% ethanol for 5 min.
    • vii
      70% ethanol for 5 min.
    • viii
      50% ethanol for 5 min.
    • ix
      Running tap water for 3 min.

      CRITICAL: Slides should remain constantly hydrated from the deparaffinization step to final mounting to avoid tissue drying leading to high background staining.

      Alternatives: Less toxic alternatives to Xylene, such as HistoChoice or CitriSolv may be used, though their efficacy in deparaffinization may vary.

      Note: Slides can be pre-heated to 60°C for 1 h prior to deparaffinization to aid in wax removal, depending on the wax content.

  • d.
    Transfer the slides to a PT Link Pre-treatment machine filled with 1X Tris-EDTA antigen retrieval buffer (pH 9.0) and heat them at 90°C for 20 min.

    • i
      After depressurization, allow slides to cool at 22°C–25°C for 1 h.

      Note: The machine can be prepared and pre-heated during the incubation intervals of deparaffinization.

      Note: Another common antigen retrieval buffer is a pH 6.0 Citrate buffer, generally preferred as it is less tissue destructive.¹⁰ However, Tris-EDTA buffer is often used for antigens that are harder to detect and is more effective at unmasking epitopes than citrate buffer. As such, a Tris-EDTA buffer is preferred in this method because of epitope masking associated with FFPE preservation. When designing a multiplex imaging panel, the same method of antigen retrieval must work for all antibodies in the panel.

      Note: If using pH 6.0 Citrate buffer, no modifications to the previous step (step 9) are needed.

      Alternatives: If a PT Link Pre-treatment machine is unavailable, a domestic pressure cooker may be used. In this approach, slides are placed in a glass container within a wire rack, heated on high for 20 min, then depressurized and allowed to cool (30 min inside the machine, 30 min outside). However, this method increases the risk of tissue detachment due to the high pressure and elevated temperatures (often exceeding 100°C). Purpose-built pathology pressure cookers may be preferred, as they maintain sub-boiling temperatures (e.g., Biocare’s Decloaking Chamber).

• 10.
  Quench tissue autofluorescence using LED photobleaching (adapted from Autofluorescence Quenching Protocol for CODEX, available here: https://www.akoyabio.com/wp-content/uploads/2020/07/Customer-Demonstrated-Protocol-Autofluorescence-Quenching-Mar2020.pdf):

  • a.
    Place the cooled slides in a glass container and fully submerge with Bleaching Solution.
  • b.
    Place the container between two LED lamps and incubate at 22°C–25°C for 45 min.
  • c.
    Discard the used solution and replace with fresh Bleaching Solution. Repeat photobleaching for another 45 min.

CRITICAL: Always use freshly prepared solution for each photobleaching step.

Note: This extended LED photobleaching step reduces autofluorescence associated with FFPE tissue staining.

Note: For tissues with high autofluorescence (e.g., heart, liver), the concentration of H₂O₂ can be increased to 10%.

Note: For fragile tissues prone to folding or detachment, perform the second LED photobleaching step in 1X PBS instead of Bleaching Solution.

• 11.Rinse the slides in distilled water and dry the glass around the tissue section with a Kimwipe.
• 12.Circle tissue with a hydrophobic PAP pen and rinse the slide in one change of ddH₂O for 2 min to ensure the hydrophobic barrier created remains intact.

Note: All staining solutions from blocking to the final mounting should be applied in a humidified chamber to prevent drying of the sample during incubation periods, to ensure optimal staining and image quality.

Note: A humidified chamber can be easily assembled from common laboratory supplies. Required materials include a container with a lid, a flat base to support the slides, paper towels, and aluminum foil or parafilm. Line the bottom of the container with folded paper towels and add water until they are damp but not excessively wet, to maintain humidity. Place a flat base (e.g., a glass plate or pipette tip rack) above the towels, then cover with a layer of parafilm to provide a flat, non-absorbent surface for slides and to preserve surface tension. Use the lid during long incubations to prevent evaporation and light-sensitive steps to prevent photobleaching. The chamber can be reused by rinsing the container and replacing the damp paper towels before each use.

Note: From this point forward, adjust solution volumes based on the tissue area outlined by the PAP pen and the number of slides being processed. As a reference, a tissue section of approximately 20 x 40 mm (800 mm²) requires around 150-200 μL to ensure complete coverage, while a smaller section of 5 x 5 mm (25 mm²) requires 40 μL or less. In most cases, 200 μL is sufficient to cover the largest sections.

• 13.Rinse with 3 changes of IF Wash Buffer for 5 min each.
• 14.Block the slides for 30 min at 22°C–25°C with Primary Blocking Buffer, then rinse with 3 changes of IF Wash Buffer for 5 min each.

Note: The quenching and blocking steps are crucial for reducing background and non-specific staining, to reduce the likelihood of falsely detecting the presence of an antigen.¹

Note: Blocking duration may require optimization depending on the tissue type and background staining. Researchers can adjust this parameter to determine the optimal blocking time for their samples.

• 15.
  Incubate tissues in primary and secondary antibody.

  Note: These steps involve applying the primary antibody to the sample, allowing it to bind to its target antigen. This step is crucial for enabling its subsequent detection through a fluorescent labeled secondary antibody. The temperature and incubation time can influence the binding efficiency of the primary antibody and are important considerations during the optimization process. The choice of fluorophore conjugated to each secondary antibody is also a critical consideration when designing and optimizing antibody panels for multiplexed imaging.¹

  • a.
    Dilute the primary antibody in Antibody Dilution Buffer and incubate slides in primary antibody in a humidified chamber at 4°C for 16 h.

    Note: For initial optimization, an approximate dilution of 1 in 100 can be used. The affinity of the antibody for the antigen affects the optimal antibody concentration. As such, staining specificity can be improved for some antibodies by incubating with a higher or lower concentration of antibody for 16 h.

    Note: For some antibodies, staining specificity can be improved by incubating the primary antibody for 30 min at 37°C or for 3 h at 22°C–25°C, instead of a 16 h incubation, as longer incubation periods can increase non-specific signal. Higher concentration of primary antibody paired with shorter incubation time at physiological temperature can help select for higher affinity binding while reducing low-affinity binding/background.

  • b.
    Rinse the slides in 3 changes of IF Wash Buffer for 5 min each.
  • c.
    Incubate slides for 1 h at 22°C–25°C with secondary antibody conjugated to a fluorophore, diluted 1 in 500 in Antibody Dilution Buffer.

    CRITICAL: Slides should remain protected from light from the application of the secondary antibody to the final imaging step to prevent photobleaching and degradation of the fluorophore.

    Note: For initial optimization, a secondary antibody conjugated to AlexaFluor647 (AF647) is recommended as the bright fluorophore can detect small amounts of protein. A different fluorophore (ATTO550 or Alexa Fluor 750 (AF750)) can be used based on both the SNR and the abundance of antigens present, whereby AF750 is preferred for high abundance, AF647 is used for low abundance, and ATTO550 for ‘medium’ (Figure 2). This is also an important consideration when choosing a specific barcode for conjugation in building an antibody panel for PhenoCycler multiplexed imaging.

    Note: When resources allow, swapping the order of secondary antibodies can help confirm that the staining patterns are consistent and independent of the sequence of application. This step is particularly useful for validating multiplexed experiments involving antibodies from overlapping species.

  • d.
    Rinse slides with 3 changes of IF Wash Buffer.
• 16.
  Counterstain, mount, and image.

  Note: Counterstaining provides a visible marker for cell nuclei to allow visualization of the target protein’s cellular localization, while mounting preserves the stained sample for microscopy. A counterstain is often required for microscope autofocus and serves as a reference channel for image registration between runs in multiplexed imaging workflows.

  • a.
    Stain tissues with prepared DAPI for 10 min at 22°C–25°C.
  • b.
    Rinse the slides in 3 changes of 1X PBS for 5 min each.
  • c.
    Rinse the slide in ddH₂O.
  • d.
    Mount coverslips onto slides with Fluoromount-G, and then allow to dry for at least 15 min.
  • e.
    Image slides using the same microscope that is used for multiplexed image acquisition.

    • i.
      Clean the glass slides gently with ethanol in case of dust, condensation, or residual mounting media prior to imaging.

      Note: As a starting point for imaging AF647-conjugated antibodies on the Axioscan 7 with a 20X objective, we use an excitation filter of 625–655 nm and an emission filter of 665–715 nm, with the LED-Module 630 nm as the light source set to 50% intensity. The excitation and emission wavelengths are set to 653 nm and 668 nm, respectively, with an exposure time of 150 ms. These values only serve as baseline settings, and parameters, particularly light source intensity and exposure time, are further adjusted depending on the antibody, tissue type, and disease state to achieve optimal imaging quality.

      Note: Acquiring the image on the same microscope used for final image acquisition with the PCF, while maintaining consistent acquisition parameters, will provide a better representation of the final staining.

      CRITICAL: To minimize photobleaching, slides should be imaged as soon as possible, considering the photostability limitations of the selected mounting medium. If slides must be stored until the next day for imaging (16 h), protect them from light. For post-imaging storage (e.g., for potential re-imaging), keep slides at low temperatures (e.g., −20°C) to reduce photobleaching risk; however, some signal loss may still occur.

      Alternatives: If antibodies are being optimized for multiplexed immunofluorescence experiments that do not use the PCF system, slides can be mounted with ProLong Diamond Antifade for imaging. When mounting with ProLong, slides are kept at 22°C–25°C, in the dark, to dry for 16 h. They can then be imaged with a fluorescent slide scanner, such as AxioScan, or a widefield or confocal microscope. It is recommended to image the slide with the same microscope that will be used in the final imaging experiment for consistency in signal evaluation.

Figure 2.

Fluorophore performance across markers

Representative images showing mouse lymphoma tumor tissue stained with (A) CD19, (B) PDL1, and (C) αSMA, and visualized with three different fluorophores (ATTO550, AF647, and AF750). Images were visualized and analyzed using QuPath.⁸ In each image, scale bar is 20 μm. Nuclei are counterstained with DAPI (blue), while the specified marker of interest is shown in white. For each image, the corresponding histogram (bottom) depicts the distribution of pixel intensities across the channel, with the purple line (left) depicting the minimum channel values, and the blue line (right) depicting the maximum channel values as visualized. The SNR is calculated by dividing the pixel intensity of the positive population by the pixel intensity of the negative population. The red box indicates the fluorophore that provides the optimal SNR profile for the given marker.

Antibody validation

Timing: Variable

Assessing the quality of antibody staining, while challenging, is essential to ensure target specificity, reproducibility, and biological relevance. Here, we present steps and tools that can be used to assess the quality of antibody staining in tissue sections.

• 17.
  Perform staining with appropriate controls to assess specificity.

  • a.
    Positive controls should express the target protein at detectable levels. Negative controls (e.g., isotype controls or knockout samples) should help confirm signal specificity.
• 18.
  Verify the localization of the target protein.

  • a.
    Confirm that the staining pattern matches the expected cellular compartment or tissue region. Compare results against known biology or published images.
• 19.Cross-validate results with alternative techniques (e.g., Western blot, RNA in situ hybridization), if possible, to support antibody specificity.
• 20.
  Check for tissue autofluorescence.

  • a.
    Tissue autofluorescence can vary depending on disease state, tissue type, fixation method, and sample preparation. Autofluorescence can impact the visualization of specific markers, hinder the specificity of the signal, and reduce the SNR, interfering with accurate interpretation.
  • b.
    Include an unstained control imaged using the same microscopy settings to assess the level of autofluorescence present. If autofluorescence is detected, consider further protocol optimization (e.g., change the fluorophore, apply an alternative quenching method, or titrate reagents).
• 21.
  Assess reproducibility.

  • a.
    Perform the assay across multiple tissue sections or on tissue microarrays.
  • b.
    Use biological replicates to evaluate consistency of staining patterns.
• 22.Consult a pathologist, when possible, to review staining patterns and assist in selecting the most appropriate antibody clones. This can be particularly helpful in the case of rare clinical samples or atypical markers where it is more difficult to discern artifacts.
• 23.
  Compare staining results as reported in the literature.

  • a.
    Evaluate if the observed staining matches published localization or expression patterns.

Note: Use publicly available gene and protein databases (see Table 2) to review reported expression patterns. These tools are useful for validating the expected distribution and abundance of the target protein.

Table 2.

Gene and protein expression databases for antibody validation

Resource name	Description	Link
Human Protein Atlas	Provides protein expression and localization data in tissues and cells	https://www.proteinatlas.org
UniProt	Comprehensive resource for protein sequence, function, and annotations	https://www.uniprot.org
NCBI (National Center for Biotechnology Information)	Contains gene and protein databases, including Gene, RefSeq, and GEO profiles	https://www.ncbi.nlm.nih.gov
CellMarker	A curated database of cell type-specific markers for human and mouse	http://bio-bigdata.hrbmu.edu.cn/CellMarker/
Atlas of Genetics and Cytogenetics in Oncology and Hematology	Includes cytogenetic, molecular, and clinical data for cancer-related genes	http://atlasgeneticsoncology.org
Expression Atlas	Gene and protein expression data across tissues, conditions, and cell lines	https://www.ebi.ac.uk/gxa/home
Human Proteome Map	Provides mass spectrometry based proteomic data across human tissues	http://www.humanproteomemap.org

Building a multiplexed imaging antibody panel

Timing: Variable

The process of building a multiplexed antibody panel generally begins with defining a biological question and prioritizing markers. General considerations such as antigen abundance, tissue type, autofluorescence, and expression patterns are outlined in the Experimental Design section above and apply broadly to both conventional immunofluorescence and multiplexed imaging platforms. This can then be followed by system-specific implementation steps (e.g., PCF, IMC, Opal staining, Multiplex IHC-IF, etc.).

Here, we extend on these principles and describe how they are implemented in the context of the PCF system. After selecting and validating antibody clones in single-plex IF, the PhenoCycler workflow requires: (1) barcode and reporter selection, (2) antibody conjugation, and (3) validation of both individual antibodies and the full multiplexed panel.

Note: For laboratories that are interested in performing routine multiplexed imaging, it is beneficial to design initial panels with broad immunophenotyping capacity. These panels can be expanded modularly as needed. Starting with a robust core panel reduces both the time and cost associated with repeated antibody validation and panel development.

• 24.
  Select barcodes and reporters for PhenoCycler-Fusion multiplexed imaging experiments.

  Note: We describe the procedure for assigning appropriate barcodes and corresponding reporters during multiplexed antibody panel design here. In the PhenoCycler system, barcodes are the unique oligonucleotide sequences conjugated to antibodies, while reporters are fluorescent dyes coupled to short oligonucleotides that are complementary to the corresponding barcode. Barcode assignment should be based on antigen abundance, tissue type, and the fluorophore that provides the optimal SNR as determined during antibody optimization.

  • a.
    Determine fluorophore performance from antibody optimization.

    • i.
      Review the images from antibody testing to identify the fluorophore that yields the highest SNR for each antibody in the target tissue.
    • ii.
      Classify each antigen as high, medium, or low abundance, as detected by the target antibody.
  • b.
    Assign fluorophores based on antigen abundance and SNR.

    • i.
      For antibodies with high antigen affinity/abundance and a low background signal, assign reporters with a higher background tolerance (e.g., AF750).
    • ii.
      For medium affinity/abundance antibodies, assign reporters that offer balanced brightness and background signal (e.g., ATTO550).
    • iii.
      For antibodies with low affinity/abundance, prioritize bright fluorophores (e.g., AF647).
  • c.
    Assign barcodes to antibodies:

    • i.
      Refer to the Akoya Biosciences’ PhenoCycler barcode index for available barcode-fluorophore pairings to select barcodes compatible with fluorophore choice.

      Note: When creating different multiplexed panels, be sure to plan barcode assignment appropriately to avoid overlap.

  • d.
    Document barcode assignments.

    • i.
      Maintain a detailed table of antibody-barcode-reporter combinations and include antigen abundance classification and staining performance notes (Figure 3).
• 25.
  Conjugate antibodies for PhenoCycler-Fusion multiplexed imaging experiments.

  Note: Antibody conjugation describes conjugating antibodies with unique oligonucleotide barcodes following antibody maleimide-thiol reduction using reagents and protocol provided by Akoya Biosciences (which can be found here: https://www.akoyabio.com/wp-content/uploads/2023/04/QRC-Conjugation-workflow_v2.pdf). This step involves reducing the antibody, conjugating it with a DNA oligonucleotide, and recovering the conjugate oligonucleotide-antibody following washing.

  • a.
    Label 50 kDa MWCO columns with the antibody and corresponding barcode.

    CRITICAL: A maximum of three antibodies should be simultaneously conjugated to reduce the risk of cross-contamination and over-reduction.

  • b.
    Add 500 μL of Filter Blocking Solution to each column and centrifuge at 12,000 x g for 2 min.
  • c.
    Carefully aspirate both the flow-through and liquid at the top of the column.

    CRITICAL: This is the only step where the liquid should be aspirated out of the column filter unit. Do not aspirate in subsequent steps, as the filter will contain the antibody.

  • d.
    Add 50 μg of the antibody to the top of the respective filter unit, at an adjusted volume of 100 μL. Centrifuge at 12,000 x g for 8 min and discard the resulting flow-through.

    Note: Confirm antibody concentration using a Nanodrop before conjugating to ensure an accurate volume is used.

  • e.
    Add 260 μL of the prepared Antibody Reduction Master Mix to the top of each filter unit and vortex for 3 s. Incubate for 30 min at 22°C–25°C.

    CRITICAL: Do not exceed 30 min to prevent irreversible antibody damage.

  • f.
    Centrifuge at 12,000 x g for 8 min and discard the resulting flow-through.
  • g.
    Add 450 μL of Conjugation Solution to the top of each filter unit. Centrifuge at 12,000 x g for 8 min and discard the resulting flow-through.
  • h.
    Prepare the Barcode:

    • i.
      Add 10 μL of molecular biology grade nuclease free water to the Barcode to resuspend the lyophilized DNA pellet.
    • ii.
      Add 210 μL of Conjugation Solution to the resuspended Barcode.
  • i.
    Add the barcode solution to the top of each filter unit and vortex for 3 s. Incubate for 2 h at 22°C–25°C.
  • j.
    Centrifuge at 12,000 x g for 8 min and discard the resulting flow-through.
  • k.
    Add 450 μL of Purification Solution to the top of each filter unit. Centrifuge at 12,000 x g for 8 min and discard the resulting flow-through.
  • l.
    Repeat the purification step above for a total of 3 purifications. After the third purification step, the filter will contain the conjugated antibody.
  • m.
    For each antibody, label a fresh tube that can hold filter units.
  • n.
    Add 100 μL of Antibody Storage Solution to each filter unit and invert the filter unit into the new collection tube. Centrifuge at 3,000 x g for 2 min.

    Note: The final antibody volume will be around 120 μL. Akoya Biosciences does not specify the expected final concentration; however, the absolute final concentration is less critical than factors such as the quality of the starting antibody, strict adherence to the conjugation protocol, proper validation of the conjugated antibody, and the subsequent dilution into the staining cocktail.

    Note: Transfer antibodies to autoclaved screw top tubes to store and minimize evaporation risk.

• 26.
  Validate custom barcode-conjugated antibodies.

  Note: This step is used to validate the conjugation of an antibody by visualizing the antibody’s heavy chain and confirming the shift in molecular weight of the conjugated antibody compared to the unconjugated antibody.

  • a.
    Cast a 10% SDS-PAGE gel and set up gel running apparatus like a typical Western blot.

    Note: This process requires 2 wells for each antibody (unconjugated and conjugated antibody) and an additional well for the protein ladder.

  • b.
    Prepare the samples:

    • i.
      Add 1 μL of unconjugated antibody to a tube with 9 μL of 1X laemmli loading dye.
    • ii.
      Add 0.5 μL of conjugated antibody to another tube with 9.5 μL of 1X laemmli loading dye.
  • c.
    Boil the samples on a heating block at 95°C for 5 min.
  • d.
    Load samples and protein ladder into a gel and run until 50 kDa is well resolved.

    Note: We typically run the gel at 90 V for 1.5 h.

  • e.
    Remove gel from cassette and place in a glass container.
  • f.
    Incubate the gel in GelCode Blue Reagent, with gentle rocking, until the solution changes color to blue.
  • g.
    Replace GelCode reagent with distilled water and rinse with gentle rocking for 20 min.

    • i.
      Wash the gel with distilled water 3 times in the same manner for 20 min each.
  • h.
    Image the bands with any imaging apparatus that can visualize Coomassie (e.g., ChemiDoc).

    Note: The antibody bands should resolve around 50 kDa. A successful conjugation is indicated by an upward shift in weight from the unconjugated (left lane) to the barcode-conjugated antibody (right lane) (Figure 4).

• 27.
  Validate staining performance of barcode-conjugated antibodies.

  Note: Conjugated antibodies must undergo quality control and titration prior to inclusion in a full PhenoCycler experiment. Validation is performed by staining tissue sections with the conjugated antibody, followed by manual application and imaging of the complementary PhenoCycler reporters. Staining specificity and intensity are then evaluated, and optimal antibody concentrations and staining conditions are determined for use in larger multiplexed panels.

  • a.
    Perform deparaffinization and antigen retrieval as described in step 9.
  • b.
    Quench tissue autofluorescence using LED photobleaching as described in step 10.
  • c.
    Rinse the tissue with 1X PBS for 5 min. Repeat for a total of 4 washes.
  • d.
    Incubate the tissue in Hydration Buffer for 5 min at 22°C–25°C.
  • e.
    Equilibrate the tissue in Staining Buffer for 20 min at 22°C–25°C.
  • f.
    Prepare the antibody solution during tissue incubation.

    • i.
      Dilute the conjugated antibody in a mix of Staining Buffer, N Blocker, G Blocker, J Blocker, and S Blocker.

      Note: Use the concentration previously optimized for the unconjugated antibody as a starting point. Further titration may be required post-conjugation.

      Note: The buffer containing blockers used for the antibody cocktail in this part of the protocol differs from the antibody dilution buffer used above in single-plex IF as the PhenoCycler workflow integrates staining and blocking into a single step.

  • g.
    Stain the tissue with the prepared antibody solution. Incubate slides in primary antibody solution in a humidified chamber at 4°C for 16 h.

    CRITICAL: Avoid introducing bubbles when applying the antibody.

    Note: Staining time and temperature may need to be re-optimized following conjugation.

  • h.
    Prepare 1X PhenoCycler buffer for use in subsequent steps. Use this solution to prepare both Hybridization and Stripping buffers.

    Note: The preparation of the Stripping buffer is exothermic; allow the solution to cool before use.

  • i.
    Perform manual hybridization and stripping cycles:

    • i.
      Incubate the slide in Hybridization buffer for 2 min at 22°C–25°C.
    • ii.
      Transfer to Stripping buffer for 5 min at 22°C–25°C.
    • iii.
      Repeat in fresh Stripping buffer for 5 min.
    • iv.
      Incubate again in Hybridization buffer for 2 min.

      Note: Use fresh solutions and clean containers (e.g., Coplin jars) for each buffer exchange.

  • j.
    Rinse slides twice in 1X PhenoCycler buffer.
  • k.
    Prepare the reporter solution.

    • i.
      Prepare 250 μL of the Reporter Stock Solution in 1X PhenoCycler buffer, with DAPI at a concentration of 1 in 300, and salmon sperm DNA in a concentration of 1 in 10.
    • ii.
      Add 5 μL of the Reporter, complementary to the Barcode, in the Reporter Stock Solution.

      Note: The Reporter used will depend on the Barcode the antibody was conjugated to. For instance, an antibody conjugated to the Barcode BX001 will require the Reporter RX001 (of the predetermined fluorophore).

  • l.
    Apply the reporter solution to the tissue for 10 min in a humidified chamber. Adjust volume based on the tissue size and number of slides.

    CRITICAL: Protect from light. Use an amber tube for the light-sensitive reporter solution.

    CRITICAL: Avoid bubbles when applying the reporter.

  • m.
    Mount the slide in 1X PBS using a number 1.5 coverslip. Protect from light and allow to dry for 15 min.
  • n.
    Image the slide using the PCF.
  • o.
    Assess staining performance (steps 17–23) to optimize antibody conditions and maximize SNR. Compare staining intensity and pattern to standard IF results to evaluate fidelity.

    Note: Perform one final validation with two additional markers: one that co-localizes with the target and one that does not. This is used to assess non-specific binding and signal interference.

    Note: Multiple antibodies and reporters can be tested simultaneously, if they are labeled with distinct fluorophores. To test multiplexing, stain with multiple antibodies and reporters in a single incubation step using their respective working dilutions. This will allow for validating the multiplexing capability of the conjugated antibodies and further optimize staining parameters.

Figure 3.

Representative table of antibody-barcode-reporter assignments with corresponding staining performance notes

This mock table illustrates how antibodies are paired with specific barcodes and reporters during panel design. For each antibody, notes on staining performance are included, such as which fluorophore yielded stronger SNR and showed optimal tissue staining patterns, and any variability across fluorophores. These tables provide a practical reference for selecting antibody-reporter pairings and for refining multiplexed panel performance during optimization.

Figure 4.

Barcode-conjugated antibody validation by SDS-PAGE

Representative gel electrophoresis showing unconjugated and barcode-conjugated antibody samples for CD8, GATA3, and PD-1. All antibodies resolve around 50 kDa, consistent with their expected molecular weight. Successful conjugation is indicated by an upward mobility shift of the barcode-conjugated antibodies compared to their unconjugated counterparts.

PhenoCycler multiplexed imaging

Timing: Variable

Once all the antibodies within the panel have been conjugated and optimized, it is time to proceed to the final staining experiment. This involves designing the reporter cycles as well as tissue staining and reporter plate preparation.

Note: Prior to final staining, each antibody must be carefully assigned to a reporter cycle. Reporter cycles refer to the iterative process of applying, imaging, and removing fluorescently labeled oligonucleotide reporters that hybridize to their complementary barcode-conjugated antibodies. Thoughtful cycle design is critical to minimize signal interference. Additionally, blank cycles must be incorporated at the beginning and end of the imaging experiment for use in background correction and image alignment.

• 28.
  Design reporter cycles using the PhenoCycler Experiment Designer software. Consider the following factors:

  • a.
    Fluorophore compatibility.

    • i.
      Each cycle can image up to three antibodies, each conjugated to barcodes detected by reporters with spectrally distinct fluorophores (ATTO550, AF647 and AF750 in this case).
    • ii.
      A single cycle must not include reporters with overlapping fluorophores.

      CRITICAL: Using two reporters with the same fluorophore in a single cycle will result in both being detected in the same fluorescence channel, making it impossible to attribute the observed signal to a specific biomarker.

  • b.
    Marker localization.

    • i.
      Group antibodies targeting markers that are not expected to co-express on the same cell type or structures (based on published literature and or previous antibody optimization steps) within the same cycle. This strategy helps aid in identifying and reducing signal spillover (Figure 5).
• 29.
  Include blank cycles within the reporter cycle design.

  • a.
    Add a “blank” cycle as the first and last cycle of each staining experiment. Blank cycles will have DAPI only, but no fluorophore-conjugated reporters.
  • b.
    Include additional blank cycles, if necessary, to help strip residual signal.

Note: Antibodies in the PhenoCycler workflow remain bound to the tissue throughout the experiment, while the reporters alone are cycled on and off. As such, it is critical to ensure that the reporters are fully stripped after each round of imaging. Blank cycles following antibodies with a propensity to retain reporters serve as a quality control step by revealing incomplete reporter removal and ensuring that the signal observed in subsequent cycles is accurate, rather than carryover from previous cycles or background noise.

Note: Our team uses DRAQ5 in the Cy5/AF647 channel, along with DAPI, to provide a secondary nuclear signal. DRAQ5 is brighter and has less background signal than DAPI.

• 30.
  Optimize imaging parameters for each marker.

  • a.
    Adjust exposure times for each reporter channel based on antibody dilution, signal intensity, fluorophore brightness, and by monitoring the imaging histogram to aim for approximately half of the dynamic range (Figure 5).

    CRITICAL: Exposure settings must remain consistent across all experimental conditions to ensure reliable comparison.

  • b.
    Use previously obtained single-plex validation data to inform exposure settings for each marker.

    Note: Careful design of reporter cycles and exposure settings is essential for generating high quality, reproducible multiplexed imaging data. Maintaining detailed documentation for each reporter-barcode-marker assignment can support downstream troubleshooting and data analysis and is important when accounting for tissue-specific parameters. For example, antibody signals such as CD45, CD8, and CD19 are typically much stronger in immune-rich tissues like lymph nodes, often necessitating shorter exposure times. In contrast, in immune depleted tumors where immune cells are rare, longer exposure times may be required. Documenting these adjustments ensures reproducibility and facilitates accurate interpretation across experiments.

• 31.
  Stain tissue and prepare reporter plate.

  Note: This portion of the protocol is adapted from the Akoya Biosciences PhenoCycler-Fusion User Guide. For more detailed steps, refer to Abraham et al.,¹ Donovan et al.,¹¹ and the Akoya Biosciences FFPE Tissue Staining and Reporter Plate Preparation guide (https://www.akoyabio.com/wp-content/uploads/2022/04/QRC-Staining-and-Reporter-Plate-preparation-FFPE.pdf).

  • a.
    Follow steps 27a–27g using all conjugated antibodies intended for multiplexed staining.

    CRITICAL: For final PCF experiments using the fluidics system, do not use a PAP pen to create hydrophobic barriers. Doing so may interfere with flow cell assembly or prevent proper fluid exchange over the tissue.

    CRITICAL: The total antibody volume should not exceed 40% of the final staining cocktail to ensure sufficient blocking.

    Note: If antibodies in the panel require different incubation times (e.g., 30 min vs 16 h), first stain with the short-incubation antibodies. Then, re-equilibrate the slide in Hydration Buffer and Staining Buffer (1 rinse each) before applying the long-incubation antibody cocktail.

    Note: Prepare separate antibody cocktails for different incubation windows.

  • b.
    Fix the tissues post-staining:

    • i.
      Incubate slide in fresh Staining Buffer for 2 min at 22°C–25°C.
    • ii.
      Transfer slide to a second aliquot of Staining Buffer for 2 min at 22°C–25°C.
    • iii.
      Apply Post-Staining Fixing Solution for 10 min at 22°C–25°C.
    • iv.
      Rinse the slide in 1X PBS, 3 times for a total of 9 washes.
    • v.
      Incubate the slide in ice-cold methanol for 5 min on ice.
    • vi.
      Rinse again in 1X PBS, 3 times for a total of 9 washes.
    • vii.
      Apply Final Fixative Solution and incubate in a humidified chamber for 20 min at 22°C–25°C.

      CRITICAL: Avoid bubbles when applying fixative.

    • viii.
      Rinse in 1X PBS, 3 times for a total of 9 washes.
  • c.
    Store the slides.

    • i.
      Submerge in Storage Buffer and store at 4°C.

      Pause point: Slides can be kept in Storage Buffer at 4°C for up to 5 days before imaging.

  • d.
    Prepare the reporter plate:

    • i.
      Prepare Reporter Stock Solution according to the number of imaging cycles.
    • ii.
      Label amber tubes for each cycle. Add 5 μL of each reporter oligo corresponding to that cycle. Complete to a volume of 250 μL with Reporter Stock Solution.

      CRITICAL: Protect all reporter solutions from light.

      CRITICAL: Mix gently by pipetting – do not vortex.

      Note: Keep reporters on ice and centrifuge for 10 s before pipetting to ensure all liquid is collected from the tube cap and walls.

    • iii.
      Pipette each reporter solution into a black-walled 96-well plate, assigning one well per imaging cycle (including blank cycles).
    • iv.
      Cover the wells with the adhesive 96-well plate seals.

      Pause point: The sealed reporter plate can be stored at 4°C for up to 2 weeks.

  • e.
    Acquire images using the PCF system.¹¹

    • i.
      Run the default PhenoCycler acquisition protocol, applying exposure settings and cycle design parameters as previously defined in steps 28–30.

Figure 5.

Example of a reporter plate design for multiplexed imaging

The representative reporter plate consists of five reporter cycles corresponding to their respective wells in a 96-well plate. Each cycle map lists the antibody, barcode, and exposure time for markers assigned to their designated fluorophores, along with the blank (DAPI only) cycles included for background correction and image alignment. All cycles include DAPI, to allow for tissue detection by the fluorescence microscope. Not all reporter cycles contain three fluorophores, reflecting intentional design considerations or availability of antibodies (i.e., the number of antibodies that are conjugated to the different fluorophores can vary). Specifically, markers that have reporters conjugated to the same fluorophore or co-localize are separated into different cycles to prevent signal overlap. This example illustrates how cycle design can be tailored to balance experimental constraints with optimal panel performance.

Expected outcomes

This protocol provides a standardized and reproducible workflow for performing and optimizing antibody-based immunofluorescence tissue histology, as well as designing and optimizing antibody panels for multiplexed imaging with a specific emphasis on the PCF platform. Upon successful completion, users can expect to generate a fully validated, custom antibody panel tailored to their tissue type and biological question. Each antibody in the panel will have been titrated and optimized for compatibility, specificity, and high SNR, ensuring robust staining performance across FFPE tissues.

In addition to the antibody panel, users will have a finalized cycle and reporter plate design with appropriate barcode and fluorophore assignments, organized to minimize signal crosstalk, reduce the risk of cross-reactivity, and incorporate blank cycles for background subtraction (Figure 5).

Validated reporter plates and stained tissue sections prepared through this protocol are expected to yield high-quality, spatially resolved protein expression data. These data are suitable for downstream analysis of cellular phenotypes, tissue architecture, and spatial relationships within the microenvironment.¹ Importantly, the optimization process also generates validated antibody conditions that can be readily applied to conventional single marker or conventional 2-4 marker IF assays, in addition to multiplex IF, thereby broadening the utility of the work beyond a single platform.

Quantification and statistical analysis

For guidance on the analysis of multiplexed imaging data, users may refer to the open-source QuPath pipeline developed by Dr. Madelyn J. Abraham¹ or consult additional analytical frameworks available in the literature.¹²

Limitations

This protocol offers a standardized framework for designing antibody panels for multiplexed imaging, but certain limitations remain.

Antibody selection and validation can be both time-consuming and costly, especially when assembling large panels. While comparing antibodies targeting the same protein from different vendors can strengthen confidence in accuracy and reproducibility, this approach substantially increases costs.¹³ Furthermore, antibodies raised against different epitopes can lead to different results depending on the accessibility of the target epitope within the sample of interest.¹³ Additionally, many antibodies are optimized for fresh-frozen tissues and often perform sub-optimally in FFPE samples, necessitating further optimization or replacement with alternative clones. Even antibodies well validated for a specific tissue and preservation method may not perform equivalently across other tissue types or disease states.

Tissue autofluorescence, a notable concern for FFPE tissues, also poses a challenge. High autofluorescence can markedly reduce SNR and compromise detection accuracy, even when photobleaching or quenching strategies are applied.

Finally, the second half of this protocol requires access to specialized instruments, such as the PCF, as well as trained personnel for imaging and data analysis. The financial burden of instrumentation, reagents, conjugation kits, and data acquisition can limit scalability. Beyond these considerations, panel development and imaging are often labor- and time-intensive, which may limit throughput. Downstream data analysis requires access to computational resources and bioinformatics expertise, whereby reproducibility may vary depending on the pipelines used.

These limitations, collectively, highlight the importance of rigorous antibody screening, repeated validation, strategic resource planning, and transparency in reporting analysis methods to ensure successful panel development and reproducible imaging results.

Troubleshooting

Problem 1

The antibody does not perform well in FFPE tissues.

Many of the antibodies that have been frequently used in the literature have been optimized for FF tissues, which could contribute to poor performance in FFPE samples.

Potential solution

Compare staining against a known positive control FFPE sample and include a negative control expected to show no signal. Alternatively, validate a new antibody by comparing performance across both FF and FFPE tissues to assess compatibility.

Problem 2

The antibody is not available carrier-free.

Potential solution

If an alternative clone is not available and the specified antibody cannot be obtained carrier free, one option is to use a biotinylated Fab secondary antibody instead of directly conjugating the primary antibody.

A Fab secondary antibody is generated by enzymatic digestion of a whole antibody and contains only the antigen-binding site (Fab region) and lacks the Fc region.¹⁴ In this workflow, the primary antibody is applied as usual, followed by a biotinylated Fab secondary antibody that recognizes the Fc portion of the primary. Streptavidin is then used to bind biotin, enabling both detection of the primary antibody and signal amplification.

This approach requires additional optimization of both primary and secondary antibody conditions (e.g., dilutions, incubation times, amplification levels, etc.). An avidin-biotin block must also be applied prior to primary antibody incubation. For integration into a multiplexed workflow, further considerations are necessary: the streptavidin cycle should be the final cycle revealed by the instrument, and there is an elevated risk of fluidics clogging. These issues may require additional blank cycles and rigorous instrument cleaning, while also hindering the possibility of reacquisition on the PhenoCycler platform in case of instrument or technical concerns.

For detailed support on adapting this strategy to multiplexed instruments, contact the corresponding author.

Problem 3

Antibody is not available as a purified protein (e.g., contains glycerol).

Potential solution

A buffer exchange can be performed to remove interfering components such as glycerol or albumin through mild IgG purification. However, it is important to note that up to 50% of antibody loss may occur. As such, for PhenoCycler experiments requiring at least 50 μg of antibody for conjugation, it is recommended to begin with a minimum of 100 μg.

This protocol can be performed using Thermo Scientific’s Zebra Desalt Spin Columns (Cat. #89889). Detailed instructions are provided here: https://documents.thermofisher.com/TFS-Assets/LSG/manuals/MAN0011529_Zeba_Spin_DesaltCol_UG.pdf.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dr. Sonia V. del Rincón, soniavictoria.delrincon@mcgill.ca.

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contacts, Dr. Madelyn J. Abraham, madelyn.abraham@mail.mcgill.ca, or Dr. Sonia V. del Rincón, soniavictoria.delrincon@mcgill.ca.

Materials availability

This study did not generate new unique reagents.

Data and code availability

This protocol does not report original datasets or code.

Acknowledgments

This work was supported by grants from the Canadian Institutes for Health Research (PJT-178194) and Cancer Research Society and Canadian Liver Foundation (award 1053451) to S.V.d.R. This research was made possible through a collaboration with the Réseau de recherche sur le cancer (RRCancer), which is financially supported by Oncopole, the FRQ cancer division, and receives funding from Merck Canada Inc.; GSK; Pfizer; and the Ministère de l’Économie, de l’Innovation et de l’Énergie du Québec (https://doi.org/10.69777/34701. RRCancer is affiliated with the Canadian Tumor Repository Network (CTRNet). V.G. was financed by the DRI EDIA Champions Award from the Digital Research Alliance. We thank Dr. Josie Ursini-Siegel for her constructive criticism and helpful comments on an earlier draft of this manuscript.

Author contributions

Conceptualization: V.G., M.J.A., C.G., and S.V.d.R.; methodology: V.G., M.J.A., and C.G.; data collection: V.G., M.J.A., and C.G.; writing – review and editing: V.G., M.J.A., and S.V.d.R.; funding acquisition: W.H.M. and S.V.d.R.

Declaration of interests

The authors declare no competing interests.