Protocol for tyramide signal amplification immunohistochemical detection of Notch1 signaling in the vascular system

Summary

Notch signaling is a pivotal regulator in the vascular system that is essential for development, angiogenesis, and maintaining vascular homeostasis. Here, we present a protocol for tyramide signal amplification (TSA) immunohistochemistry, tailored explicitly for detecting Notch signaling components in vascular tissues. We describe steps for utilizing tailored antigen retrieval techniques, specific blocking solutions, and a complex of avidin/biotin-horseradish peroxidase conjugate with tyramide, along with optimized washing steps.

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

Graphical abstract

Highlights

• •Instructions for preparing various vascular tissues to detect activated Notch1 signaling
• •Steps for antigen retrieval to enhance the accessibility of antigens
• •Guidance on secondary signal amplification using tyramide signal amplification

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

Notch signaling is a pivotal regulator in the vascular system that is essential for development, angiogenesis, and maintaining vascular homeostasis. Here, we present a protocol for tyramide signal amplification (TSA) immunohistochemistry, tailored explicitly for detecting Notch signaling components in vascular tissues. We describe steps for utilizing tailored antigen retrieval techniques, specific blocking solutions, and a complex of avidin/biotin-horseradish peroxidase conjugate with tyramide, along with optimized washing steps.

Before you begin

Conventional immunofluorescence typically involves fluorescently labeled antibodies binding to target antigens, with specific fluorescent signals observed through fluorescence microscopy. In contrast, TSA utilizes the catalysis of horseradish peroxidase (HRP) to activate fluorophore-conjugated tyramide in the presence of hydrogen peroxide (H₂O₂). The activated tyramide covalently binds to nearby tyrosine residues, enhancing the fluorescent signal. TSA offers several advantages over conventional immunofluorescence, including increased sensitivity, multi-label capability, improved operability, and a broader range of applications, particularly for detecting low-abundance targets and enabling multiple labeling.² For further details, please refer to Table 1.

Table 1.

Comparison between TSA and conventional immunofluorescence

Aspect	TSA	Conventional immunofluorescence
Applications	Low-abundance proteins	High-abundance proteins
Multiplexing Capabilities	More fluorescent labels	A limited number of colors
Antibody Species	Same species allowed	Different species required
Specificity	High	Relatively low
Sensitivity	High	Relatively low

Institutional permissions

Mice are used in this protocol. All mouse experiments and procedures follow the protocol approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Tenth People’s Hospital.

Prepare retinal whole mount

Timing: 14 h

This step involves the dissection and fixation of the retina, ensuring its preservation for detailed analysis.

• 1.Euthanize murine pups at postnatal day 5 (P5). Fix the eyes in 4% PFA in PBS for 2 h on ice.³
• 2.Dissect the retinas from the eyes carefully. Following initial fixation, the retinas were further fixed in 100% methanol at −20°C for 12 h.

Prepare en face aorta

Timing: 1 h

This step outlines the preparation of the en face aorta.

• 3.Euthanize mice at P30. Dissect and fix the aorta in 4% PFA in PBS for 10 min at 25°C.⁴
• 4.Remove the adventitial tissues to expose the underlying structure carefully.
• 5.Cut the aorta longitudinally to expose the endothelium, facilitating further processing for staining.

Human umbilical vein endothelial cell culture and stimulation

Timing: 16 h

Human umbilical vein endothelial cells (HUVECs) are cultured and stimulated by DLL4 ligands.

• 6.Culture HUVECs in the endothelial cell medium (ECM).
• 7.Coat the glass bottom cell culture dish with immobilized DLL4 ligands (1.5 μg/mL) in 1× PBS at 37°C for 4 h.
• 8.Remove the buffer and plate the cells; allow them to adhere in 37°C 5% CO₂ cell culture incubator for 12 h.
• 9.Fix the cells with 4% paraformaldehyde (PFA) in PBS at 25°C for 10 min.⁵

Note: Following the stimulation of DLL4 ligands, robust Notch signaling activity was observed, as demonstrated in Figure 3.

Figure 3.

Localization of NICD in cultured HUVECs with Notch ligand stimulation

Endothelial cells incubated with the Notch1 ligand DLL4 show enhanced Notch signaling activity, as evidenced by stronger NICD staining (red), compared to HUVECs cultured without the recombinant human DLL4 ligand (rhDll4).

Prepare cardiac and pulmonary tissues

Timing: 16 h

Cardiac and pulmonary tissues are prepared for paraffin sectioning to facilitate subsequent staining and analysis.

• 10.Euthanize murine pups at P1 and P5. Dissect and fix the heart and lungs in 4% PFA in PBS for 12 h at 4°C.^6,7
• 11.Dehydrate the tissues using a graded ethanol series, clear them in xylene, embed them in paraffin, and prepare sections for further analysis.

Note: We performed TSA staining on P1, P5, and P30 mice, utilizing both male and female littermates, and obtained consistent results.

Note: The exposure time of a sample to the fixative is critical and must be optimized based on factors such as tissue density, penetration rate, and temperature. In our study, we have implemented specific fixation protocols tailored for various samples, as detailed in our guide on tissue fixation (https://www.ndbbio.com/post/tissue_fixation).

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
NICD (1:200)	CST	4147S
JAG1 (1:200)	CST	2620S
ERG (1:200)	Abcam	ab92513
IB4 (1:100)	Sigma	L2895
CDH5 (1:200)	BD Pharmingen	555661
CDH5 (1:200)	BD Pharmingen	555289
Endomucin (1:100)	Santa Cruz	SC-65495
αSMA-CY3 (1:500)	Sigma	C6189
Alexa Fluor 488 (donkey anti-rat)	Jackson ImmunoResearch	721-545-150
Alexa Fluor 488 (donkey anti-rabbit)	Jackson ImmunoResearch	711-545-152
Chemicals, peptides, and recombinant proteins
Paraformaldehyde (PFA)	Sigma	P6148
Phosphate buffer saline (PBS)	Yeasen (China)	41403ES76
Methanol	Sangon (China)	A506806-0500
Ethanol	Sangon (China)	A500737-0500
Xylene	Sangon (China)	A530011-0500
Hydrochloric acid (37%)	Sigma	258148
Sodium citrate tribasic dehydrate	Sigma	S4641
Hydrogen peroxide (H2O2)	Sigma	216763
Tween 20	Yeasen (China)	60305ES76
Triton X-100	Sigma	T8787
Bovine serum albumin (BSA)	BBI (China)	A600332-0100
Magnesium chloride (Mgcl2)	Sigma	63069
Donkey serum	Jackson ImmunoResearch	017-000-121
F(ab)2 fragment (donkey anti-rabbit)	Jackson ImmunoResearch	711-036-152
TSA Plus Cyanine3 system (1:100)	PerkinElmer	NEL744001KT
TSA Plus fluorescein system (1:100)	PerkinElmer	NEL741001KT
DAPI	Invitrogen	D1306
Recombinant DLL4-His	R&D Systems	1506-D4
Anti-Rabbit IgG (biotinylated)	Vector Laboratories	BA-1000-1.5
ABC kit	Vector Laboratories	PK-6100
Mounting medium	Sigma	F4680
Experimental models: Organisms/strains
C57BL/6 mice (M/F; post-natal day 1, 5, 30)	Charles River	N/A
Experimental models: Cell lines
HUVECs	ScienCell	8000
ECM	ScienCell	1001
Others
Sharp dissection scissors	GEUDER AG (China)	G-19745
Student Vannas spring scissors	Fine Science Tools	91500-09
Forceps	Dumont (China)	0208-5-PS
Microscope slides	Servicebio (China)	G6012-1
Microscope cover glass	Fisher Scientific	12-541-012
35 mm dish (glass bottom)	Fisher Scientific	NC9341562
PAP pen	Vector Laboratories	H-4000
Fluorescence microscope	Olympus	IX83
Confocal microscope	Nikon	TI2-E+A1 R

Materials and equipment

Antigen retrieval citrate buffer

Diluting hydrochloric acid

Dilute 4.93 mL of 37% hydrochloric acid (HCl) with distilled water to a final volume of 50 mL, resulting in a 1 M HCl solution.

Store at 25°C. Preferably use within one month.

10 mM sodium citrate buffer

Weigh 2.94 g of Tri-sodium citrate dihydrate into a sterile beaker containing 1 L of distilled, deionized water (ddH₂O). Use a magnetic stirrer to mix the solution until the Tri-sodium citrate is completely dissolved. This forms a 10 mM sodium citrate buffer solution with a pH (∼ or approximately) 6.0 by supplementing the buffer with 3 mL 1 M hydrochloric acid (HC). This eliminates the need for frequent pH adjustments.

Store at 25°C. Preferably use within one week.

Diluting Tween 20

Dilute 0.3 mL of Tween-20 to a final volume of 1000 mL with PBS, resulting in a 0.03% Tween-20 solution.

Prepare right before use.

HistoBlocking solution I

Reagent	Final concentration	Amount
Donkey serum	5%	2.5 mL
BSA	1%	0.5 g
1 M MgCl2	20-mM	50 μL
Tween-20	0.3%	150 μL
PBS	N/A	Adjust to 50 mL
Total	N/A	50 mL

Prepare right before use.

HistoBlocking solution II

Reagent	Final concentration	Amount
Donkey serum	5%	2.5 mL
BSA	1%	0.5 g
1 M MgCl2	20-mM	50 μL
TritonTM X-100	0.3%	150 μL
PBS	N/A	Adjust to 50 mL
Total	N/A	50 mL

Prepare right before use.

DAPI solution

Dilute the DAPI stock solution 1:5000 in 1× PBS to obtain a working solution.

Prepare right before use.

Step-by-step method details

Antigen retrieval

Timing: 1 h

Samples undergo antigen retrieval using a 10 mM sodium citrate buffer to enhance the accessibility of antigens for subsequent staining procedures.

• 1.
  For antigen retrieval of retina and aorta samples:

  • a.
    wash the samples thoroughly in 1× PBS.
  • b.
    place the retina and aorta specimens in 2 mL flat-bottom Eppendorf tubes filled with 10 mM sodium citrate solution.
• 2.
  For antigen retrieval of HUVECs：

  • a.
    wash the HUVECs thoroughly in 1× PBS.
  • b.
    fill a 35 mm glass bottom dish with 10 mM sodium citrate solution.
• 3.Place all these specimens in a water bath maintained at 95°C for 15 min.

Note: Elevated temperatures and vapor can potentially damage or distort the delicate morphology of tissue samples. Conversely, omitting the heat retrieval step can lead to weak signals and increased background noise. Water-bath heating is recommended for optimal results. Water-bath heating provides precise temperature control, which is crucial for effective antigen retrieval while minimizing the risk of tissue damage, particularly for delicate samples. Previous studies have demonstrated that this method is highly effective, maintaining tissue integrity and staining quality compared to microwave or pressure cooker methods.^8,9

• 4.
  For antigen retrieval of heart and lung tissue samples：

  • a.
    deparaffinize the paraffin-embedded tissue sections by immersing them in a series of xylene bath.
  • b.
    rehydrate the paraffin-embedded tissue sections through a graded ethanol series.
  • c.
    heat the tissue samples in a microwave oven for 15 min (three cycles of 5 min each) in a 10 mM sodium citrate buffer.
• 5.Allow the antigen retrieval buffer to cool to 25°C to avoid thermal shock after heat-induced epitope retrieval.
• 6.Wash the samples twice in distilled water and 1× PBS for 5 min each.

Quenching endogenous peroxidase activity

Timing: 1 h

This step employs a 1% H₂O₂ solution to quench endogenous peroxidase activity in the samples, thereby reducing background staining and enhancing the quality of the subsequent analysis.

• 7.Incubate the samples with 1% H₂O₂ solution in 100% methanol for 40 min at 25°C.
• 8.Wash the samples in 1× PBS three times (5 min each).
• 9.Wash the samples in 0.3% Triton X-100/PBS once.

CRITICAL: Culturing cells or tissues may contain endogenous peroxidases, particularly in highly vascularized areas. An amplification reagent, such as tyramide, can lead to interactions with these endogenous peroxidases, resulting in a non-specific background signal.

Blocking

Timing: 1 h

Here, non-specific binding sites in the samples are blocked using Histoblock solution.

• 10.Incubate monolayer cell samples, including en face aorta and HUVECs, in Histoblock solution type I.
• 11.Incubate retina, heart, and lung tissue samples in Histoblock solution type II.
• 12.Carry out the incubation for 1 h at 25°C with gentle rocking to ensure even distribution and effective blocking of non-specific binding sites.

Primary antibody labeling

Timing: 16 h

This step facilitates the labeling of target proteins with a primary antibody.

• 13.Dilute the primary antibody in the appropriate Histoblock solution.

Note: For en face aorta and HUVEC samples, Histoblock solution type I was used. For retina, heart, and lung samples, which were prepared as mentioned in the blocking section, Histoblock solution type II was employed.

• 14.Discard the blocking solution via pipetting.
• 15.Add the diluted primary antibody to the samples, ensuring complete coverage.
• 16.Incubate the samples for 12 h at 4°C with gentle rocking to facilitate optimal antibody binding.
• 17.Discard the diluted primary antibody via pipetting.
• 18.Wash all samples twice in 1× PBS, with each wash lasting 5 min.
• 19.Wash the monolayer endothelial cell samples, including en face aorta and HUVEC samples, with 0.1% Triton X-100 in PBS twice for 5 min each.

Note: This washing step allows to permeabilize the cells and remove non-specifically bound antibodies.

• 20.Wash the retina and paraffin-embedded tissue sections from the heart and lung with a more concentrated solution of 0.3% Triton X-100 in PBS twice for 5 min each.

Note: This higher detergent concentration was necessary to permeabilize the thicker tissue sections and achieve thorough washing effectively.

Secondary antibody labeling and biotin amplification

Timing: 3 h

This major step enhances signal detection by allowing the biotinylated secondary antibody to bind to primary antibodies, thereby amplifying the biotin signal with an ABC kit.

• 21.Incubate the samples with a biotinylated goat anti-rabbit antibody (1:100 dilution) for 1 h on a shaker at 25°C. The antibody was diluted in a 1% BSA solution prepared in 1× PBS.

Note: This incubation step allowed the biotinylated secondary antibody to bind to any rabbit-derived primary antibodies present in the samples, facilitating the subsequent detection of the target antigens using the avidin-biotin-peroxidase complex.

• 22.
  Prepare the VECTASTAIN ABC Reagent working solutions during the secondary antibody incubation period.

  • a.
    add two drops (approximately 50 μL) of REAGENT A (gray label) 2.5 mL of buffer (PBS) in the ABC Reagent large mixing bottle.
  • b.
    add two drops (approximately 50 μL) of REAGENT B (gray label) to the same mixing bottle.
  • c.
    allow the prepared VECTASTAIN ABC Reagent to stand approximately 30 min before use.

Note: The contents of the bottle were immediately mixed thoroughly.

• 23.Add a complex of avidin/biotin-horseradish peroxidase conjugate (ABC Reagent working solutions) to the samples and incubate for 1 h at 25°C after washing in 1× PBS twice for 5 min each.
• 24.Wash all samples in 1× PBS twice for 5 min each after ABC Reagent working solutions incubation.
• 25.Wash the monolayer endothelial cell samples (en face aorta and HUVECs) with 0.1% Triton X-100 in 1× PBS twice for 5 min each.
• 26.Wash the retina and paraffin-embedded tissue sections from the heart and lung with a more concentrated solution of 0.3% Triton X-100 in 1× PBS for 5 min each.

CRITICAL: It is crucial to note that after washing with Triton X-100 solutions, further washing with PBS should be avoided. This step has been shown to significantly reduce background noise, thereby enhancing the specificity and clarity of the staining.

Tyramide signal amplification

Timing: 16 h

Tyramide Signal Amplification using Cyanine3 or Fluorescein is employed for secondary signal amplification, enabling additional double or triple immunostaining to enhance detection sensitivity.

• 27.Treat the samples with Tyramide Signal Amplification-Cyanine3 or Fluorescein (1:100 dilution) and incubate them at 25°C for 5 min.
• 28.Wash all samples twice in 1× PBS, with each wash lasting 5 min.
• 29.Wash the monolayer endothelial cell samples (en face aorta and HUVECs) with 0.1% Triton X-100 in 1× PBS twice (5 min each).
• 30.Wash the retina and paraffin-embedded tissue sections from the heart and lung with a more concentrated solution of 0.3% Triton X-100 in 1× PBS twice (5 min each).
• 31.
  The following steps are performed for additional double or triple immunostaining. Prepare the second primary antibody using the following specific blocking solution based on the sample type.

  • a.
    en face aorta and HUVEC samples: 0.3% Tween and 5% donkey serum in PBS.
  • b.
    retina samples: 0.3% Triton X-100 and 5% donkey serum in PBS.
  • c.
    paraffin-embedded heart and lung tissue sections: 0.1% Triton X-100 and 5% donkey serum in PBS.
• 32.Incubate the samples with the prepared second primary antibody solution, ensuring thorough coverage.
• 33.Incubate the samples for 12 h at 4°C with gentle rocking to promote optimal binding of the primary antibody.
• 34.Wash the samples with 1× PBS twice.
• 35.Apply the appropriate secondary antibody, selected based on the specific primary antibody used.

Note: If the second primary antibody originated from the same species as the first primary antibody, such as rabbit-derived, the samples needed to be incubated with donkey anti-rabbit F(ab')₂ fragments. This step was performed for 2 h at 25°C to minimize crosstalk between the following primary antibodies. This additional step was not applied to samples treated with primary antibodies not originating from the same species, such as the Rat anti-endomucin antibody. After this step, the samples were re-blocked with the above suitable blocking solution and incubated for 12 h at 4°C with the rabbit anti-ERG primary antibody.

Nuclear counterstaining

Timing: 20 min

Nuclear counterstaining of the samples is performed to enhance visualization of cellular nuclei.

• 36.Incubate the samples with the prepared DAPI solution for 5 min at 25°C.
• 37.Wash the samples twice in 1× PBS by gentle rocking, with each wash lasting 5 min.

Mounting and preservation

Timing: 45 min

The samples are mounted and preserved for further analysis.

• 38.Remove any excess 1× PBS from the samples.
• 39.Mount coverslips using a suitable anti-fade mounting medium and invert them onto glass slides.
• 40.Allow to dry for 30 min, then store slides at 4°C.

Imaging

Timing: 1 h

Collection of images from the stained and mounted samples facilitates visualization and analysis.

• 41.Take images of the stained and mounted samples using either fluorescence or confocal microscopy.

Expected outcomes

This study’s modified TSA staining protocol offers a rapid, cost-effective, and reliable approach for visualizing Notch1 signaling in various vascular tissues. The Cleaved Notch1 (Val1744) antibody detects the Notch1 intracellular domain (NICD) following its release by cleavage between Gly1743 and Val1744. This cleavage event signifies the activation of the Notch1 signaling pathway, allowing NICD to translocate to the nucleus and initiate the transcription of target genes.^10,11,12 Detecting NICD using this antibody is crucial for studying the dynamics and implications of Notch1 signaling in various biological processes and diseases.

In this study, we utilized a water-bath heating method for antigen retrieval for more fragile and sensitive tissue samples, including the retina, en face aorta, and HUVECs. This approach allowed for precise temperature control and effective antigen unmasking while minimizing the risk of tissue damage. This adjustment shortened the overall procedure and better preserved the morphology of these delicate tissues, as evidenced by the clear identification of NICD within the murine retinal vessel in Figure 1. The ability to effectively visualize NICD in the retinal vessels is aligned with our previous research findings.^1,13 We also demonstrated massive endogenous expression of the Notch1 Intracellular Domain (NICD) in the en face aorta samples, as evidenced by co-staining with the endothelial cell membrane marker VE-cadherin (Figure 2). HUVECs are widely used to investigate the Notch signaling pathway. Immobilized DLL4 ligands commonly activate the Notch1 receptor in cultured endothelial cells. However, fewer studies have demonstrated the localization of the cleaved, active form of the NICD with traditional immunostaining approaches. Conventional immunofluorescence does face challenges, including weak signals, limited detection of low-abundance targets, and a restricted number of labels. This study showed a significant increase in NICD levels within the endothelial cell nuclei after DLL4 ligand stimulation, as expected (Figure 3).

Figure 1.

Localization of NICD and endothelial cells in the retinal vasculature

This figure demonstrates the improved TSA immunostaining protocol, which enabled the simultaneous detection of the Notch1 Intracellular Domain (NICD, shown in red) and the endothelial cell marker IB4 (shown in green) in the retinal vessels of mice at postnatal day 5 (P5). Artery, a; Vein, v.

Figure 2.

En face visualization of Notch signaling in the mouse descending aorta

Triple immunostaining of NICD (shown in red) and the vascular endothelial cell marker VE-cadherin (VECAD, shown in green) in the aorta of adult (6-week-old) male C57BL/6 mice. The nuclei are counterstained with DAPI (blue).

Our optimized protocol demonstrated consistent and reproducible results across various tissue types. As shown in Figure 4A, our protocol enabled the visualization of NICD distribution within the vascular system of the lung tissue. The NICD signal merged with the endothelial nuclear marker ERG, confirming the presence of active Notch signaling within the endothelial cell population of the lung vasculature (Figure 4B). These findings are consistent with our previous report,⁶ contributing to our further understanding of Notch signaling roles in regulating vascular endothelial cell biology. Furthermore, our protocol effectively stained JAG1 (the main Notch1 ligand) in the lung’s vascular system. Interestingly, JAG1 was detected primarily in the vascular smooth muscle cells, with a relatively lower expression in the endothelial cells (Figures 5A and 5B). In addition to the lung tissue, the successful application of this method to heart tissues confirmed the presence of NICD in endothelial cells, as shown in Figure 6. In contrast, using the traditional immunostaining method, NICD failed to localize effectively within the retinal vessels and other cell types (Figure S1). The consistency and robustness of our modified TSA staining technique across different tissue types underscore its effectiveness for visualizing Notch signaling throughout the vascular system.

Figure 4.

Spatial distribution of activated Notch1 signaling in the murine neonatal lung

(A and B) The co-localization of the Notch Intracellular Domain (NICD, red) and or the endothelial cell marker ERG (green) in the lung sections, with nuclei counterstained by DAPI (blue). This indicates the presence of active Notch signaling within the vascular endothelial cells of the developing lung.

Figure 5.

Expression of Notch ligand Jagged-1 in the murine lung

(A) The triple immunostaining of the Notch ligand Jagged-1 (JAG1, red) and the endothelial cell marker ERG (green), also with DAPI nuclear counterstaining (blue).

(B) The co-staining of JAG1 (green) and the vascular smooth muscle cell marker α-SMA (α-Smooth Muscle Actin, shown in red), with DAPI nuclear counterstaining (blue), reveals that JAG1 is predominantly localized in the vascular smooth muscle cells of the P5 murine lung.

Figure 6.

Immunostaining of NICD and Endomucin in the murine neonatal heart at postnatal day 1

The co-localization of the NICD (red) and the specific endothelial marker Endomucin (EMCN, green) signals indicate the presence of active Notch signaling within the vascular endothelial cells of the developing murine heart at this early postnatal timepoint.

Limitations

TSA staining serves as a valuable methodology for elucidating Notch1 signaling and its implications in vascular biology and associated diseases, particularly regarding the activated Notch1 intracellular domain (NICD) and its ligand JAG1. However, this technique presents certain limitations. Notably, not all antibodies are compatible with TSA staining; for example, we could not identify a suitable antibody for effectively mapping another main Notch1 ligand, Delta-like 4 (Dll4), in vascular tissues. Therefore, additional testing of Notch-related antibodies is essential for a more comprehensive analysis.

Troubleshooting

Problem 1

Weak or absent Notch1 signaling instead of high background noise.

Potential solution

• •The appropriate antibodies, such as the Cleaved Notch1 (Val1744) antibody listed in the table, should be utilized for detecting the Notch1 intracellular domain (NICD).
• •Avoid using microwave heating-based antigen retrieval for delicate tissues, such as en face aorta, and retina (step 3).
• •The quenching of endogenous peroxidases with H₂O₂ pretreatment helps to ensure the specificity of the signal obtained during the subsequent staining or detection procedures by eliminating the potential for non-specific interactions and background noise (step 7).
• •After washing with Triton X-100 solutions before incubation with Tyramide Signal Amplification-Cyanine3 or Fluorescein, additional washing with PBS should be avoided (step 25, 26).

Problem 2

The second primary antibodies were derived from the same species which had previously undergone amplification by TSA staining.

Potential solution

• •If the second primary antibody originated from the same species as the first, such as rabbit-derived, the samples needed to be incubated with donkey anti-rabbit F(ab')₂ fragments (step 35).
• •This step was performed for 2 h at 25°C to minimize crosstalk between the following primary antibodies (step 35).

Problem 3

Excessive impurities in fluorescence images.

Potential solution

Make sure to filter the blocking solution with a 0.22 μm sterile filter before adding of detergent (step 10, 11).

Problem 4

The immune signaling from the second primary antibody was weak following NICD amplification using TSA staining.

Potential solution

• •Using the suggested specific blocking solution based on the sample type is recommended.
• •Wash the samples with 1× PBS instead of detergent (step 34).

Problem 5

The retina sample curled during mounting.

Potential solution

Cut the fixed retina into petal shapes and flatten it before incubating it with the primary antibody (step 1, 2).

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Guofu Zhu (guofu_zhu@tongji.edu.cn).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Guofu Zhu (guofu_zhu@tongji.edu.cn).

Materials availability

The materials used and generated in this study are available from the lead contact upon reasonable request with a completed Materials Transfer Agreement.

Data and code availability

This protocol does not generate any datasets or code.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (grant 82200446 to G.Z. and grant 82170521 to W.C.). Figures in the graphical abstract were created with BioRender.com.

Author contributions

Y.L., S.S., and C.X. performed the immunostaining experiments, analyzed the data, and wrote the manuscript. Z.W. and C.F. conducted the animal experiments. D.J., L.L., and W.L. provided technical assistance. W.C. and G.Z. contributed to editing the manuscript.

Declaration of interests

The authors declare no competing interests.