Immunofluorescence protocol for localizing protein targets in brain tissue from diverse model and non-model mammals

Summary

Previous immunostaining protocols are highly specific for model organisms and often not suitable for diverse specimens that are non-perfused and over-fixed (i.e., tissues sitting in fixatives for months/year). Here, we present an immunofluorescence protocol for localizing protein targets in brain tissue from 11 model and non-model mammals. We describe preparation of both fresh and fixed tissues including steps for deparaffinization, fixation, and cryoprotection. We then detail immunofluorescence procedures including antigen retrieval, reducing autofluorescence, nuclear staining, mounting, and image collection.

Graphical abstract

Highlights

• •Immunostaining technique to stain brain tissue
• •Technique suitable for a diverse set of model and non-model mammalian species
• •Technique applicable on a wide variety of brain tissue preparations

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

Previous immunostaining protocols are highly specific for model organisms and often not suitable for diverse specimens that are non-perfused and over-fixed (i.e., tissues sitting in fixatives for months/year). Here, we present an immunofluorescence protocol for localizing protein targets in brain tissue from 11 model and non-model mammals. We describe preparation of both fresh and fixed tissues including steps for deparaffinization, fixation, and cryoprotection. We then detail immunofluorescence procedures including antigen retrieval, reducing autofluorescence, nuclear staining, mounting, and image collection.

Before you begin

Preparation of the samples

Researchers have often little to no control on the preparation protocols for tissues obtained from collaborators or from brain banks. Tissues treated with different fixation and/or embedding methods usually require different immunofluorescence protocols.^1,2,3 This could lead to variabilities in the data comparison across specimens. In this section, we provide details to fix and embed tissue previously processed in different ways (i.e., fresh, formalin/paraformaldehyde-fixed, or paraffin-embedded), in order to make them consistent for the beginning of the immunostaining procedure. Proceed with the proper embedding protocol, according to the type of the sample that needs to be processed for immunofluorescence.

Note: This protocol is set for 40 μm-thick, free-floating slices, but can also be adapted to lower thickness and/or sections attached on glass slides (troubleshooting 2 and 3).

Institutional permissions

All experiments conform to the safety guidelines at SISSA. Tissues have been donated from collaborators or have been obtained from the Netherlands Brain Bank. The work on human post-mortem brains has been approved by SISSA ethical committee. Please, notice that you will need to acquire permissions from the relevant institutions prior to obtain and work on specific samples.

A. Fresh tissues

Timing: 3 days

Timing: 10 min + incubation overnight (for step 1)

Timing: 2 days of incubation + 30 min embedding (for step 2)

Timing: 1 h (for step 3)

• 1.Tissue fixation.

Incubate the tissue in either PARAFORMALDEHYDE 4% in PBS or in FORMALIN (i.e., aqueous solution of 37% formaldehyde), overnight at +4°C.

• 2.
  Tissue embedding.

  • a.
    After fixing your sample, incubate the tissue in 30%-sucrose in PBS (see materials and equipment) for at least two days (or as long as it takes for the tissue to sink to the bottom of the tube), at +4°C, for cryoprotection (troubleshooting 1).

    CRITICAL: It is important to avoid long storage of the tissues in the sucrose solution to avoid the formation of mold.

  • b.
    After having cryoprotected the tissue, fill the bottom of a cryo-mold with OCT Cryo-embedding medium (see the key resources table), lay the tissue on it and cover it with OCT, avoiding bubbles.
  • c.
    Leave the cryo-mold with the embedding tissues on a flat support either on dry ice (into the hood) or at –80°C for a few minutes, until the embedding medium solidifies.
  • d.
    Store the tissue at −80°C.
• 3.
  Tissue cryo-sectioning at the cryostat.

  • a.
    Prepare a 6-well plate filled with 2 mL of anti-freezing medium (see materials and equipment) per each well.
  • b.
    Leave the cryo-molded tissue and the 6-well plate filled with anti-freezing medium in the cryostat (at −20°C) for 30 min for acclimatization.
  • c.
    Cut the tissue sections at 40 μm thickness (troubleshooting 2 and 3) and put them in the 6-well plate filled with the anti-freezing medium. The number of sections per well depends on the section size (usually no more than 4).
  • d.
    Store the 6-well plate at −20°C.

B. formalin or paraformaldehyde fixed tissues

Timing: 2 days

• 4.If your sample is already fixed, proceed with embedding as in steps 2–3 from section A.

C. paraffin embedded tissues

Timing: 3 days

Timing: 3 h (for step 5)

Timing: 2 days (for step 6)

• 5.
  Deparaffinization of the sample.

  • a.
    Detach the paraffin-embedded sample from the plastic support and trim as much paraffin as possible all around the sample with a scalpel (this step will save you some time, but be careful to not damage the tissue).
  • b.
    Incubate the paraffin-embedded specimen in abundant xylene (in a chemical hood) until all paraffin is melted (if necessary, change the xylene every hour).
  • c.
    Re-hydrate the tissue with solutions with decreasing ethanol concentrations, as follows:

    • i.
      Ethanol 100% for 15 min.
    • ii.
      Ethanol 96% for 15 min.
    • iii.
      Ethanol 75% for 15 min.
    • iv.
      Ethanol 50% for 30 min.
    • v.
      PBS 1× for 30 min.
  • d.
    For a complete paraffin elimination, heat the antigen retrieval buffer solution (see materials and equipment) until boiling. Insert the tissue into the solution and keep it at high temperature (not inferior to 70°C) for 5 min (troubleshooting 4). Then, let the antigen retrieval buffer cool down at room temperature, before proceeding to the next steps.

CRITICAL: this step is important to avoid thermal shock.

• 6.Fixation and embedding.

Fixation is again recommended before embedding the tissue. Proceed as in section A (steps 1,2,3).

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Rabbit pAb to ALDH1L1 (dilution 1:500)	Abcam	Cat#ab190298 RRID:AB_2857848
Mouse mAb Clone:5C10 to GFAP (dilution 1:500)	Abcam	Cat#ab190288 RRID:AB_2747779
Rabbit Anti-S100 beta antibody [EP1576Y] - astrocyte marker (dilution 1:300)	Abcam	Cat#ab52642 RRID:AB_882426
Donkey anti-mouse IgG (H + L) highly Cross-Adsorbed secondary antibody, Alexa Fluor™ 488 (dilution 1:400)	Invitrogen	Cat#A-21202 RRID:AB_141607
Donkey anti-mouse IgG (H + L) highly Cross-Adsorbed secondary antibody, Alexa Fluor™ 594 (dilution 1:400)	Invitrogen	Cat#A-21203 RRID:AB_141633
Donkey anti-Rabbit IgG (H + L) highly Cross-Adsorbed secondary antibody, Alexa Fluor™ 488 (dilution 1:400)	Invitrogen	Cat#A-21206 RRID:AB_141633
Donkey anti-rat IgG (H + L) highly Cross-Adsorbed secondary antibody, Alexa Fluor™ 594 (dilution 1:400)	Invitrogen	Cat#A-21209 RRID:AB_2535795
Biological samples
Prefrontal cortex from Bos taurus (cattle)	Collaborators	N/A
Prefrontal cortex from Carollia perspicillata (Seba’s short-tailed bat)	Collaborators	N/A
Prefrontal cortex from Homo sapiens (human)	The Netherlands Brain Bank	N/A
Prefrontal cortex from Macaca fascicularis (crab-eating macaque)	Collaborators	N/A
Prefrontal cortex from Macaca mulatta (Rhesus macaque)	Collaborators	N/A
Prefrontal cortex from Mus musculus (mouse)	Collaborators	N/A
Prefrontal cortex from Pan troglodytes (chimpanzee)	Collaborators	N/A
Prefrontal cortex from Panthera leo (lion)	Collaborators	N/A
Prefrontal cortex from Panthera pardus (leopard)	Collaborators	N/A
Prefrontal cortex from Panthera tigris (tiger)	Collaborators	N/A
Prefrontal cortex from Tursiops truncatus (common bottle-nose dolphin)	Collaborators	N/A
Chemicals, peptides, and recombinant proteins
PBS tablets	Thermo Fisher	Cat#18912014
Killik O.C.T. Compound embedding medium for cryostat	Bio-Optica Milano S.p.A.	Cat#059801
Donkey serum	Sigma-Aldrich	Cat#D9663
Sudan Black B	Sigma-Aldrich	Cat#199664
Triton™ X-100	Sigma-Aldrich	Cat#T9284
TWEEN® 20	Sigma-Aldrich	Cat#P1379
Sodium citrate tribasic dihydrate	Sigma-Aldrich	Cat#71402
Fisher Chemical Permount Mounting Medium	Fisher Scientific	Cat#SP15-500
NeuroTrace™ 435/455 blue fluorescent Nissl stain	Thermo Fisher	Cat#N21479
DAPI 4′,6-diamidino-2-phenylindole dihydrochloride	Sigma-Aldrich	Cat#32670

Materials and equipment

Antifreeze medium

Reagent	Final concentration	Amount
Sucrose	30%	150 gr
Phosphate buffer saline (PBS)	0.1 M	200 mL
Ethylene glycol	30%	150 mL
Total		500 mL

Store at room temperature for a short period or at 4° for longer period.

CRITICAL: Ethylene glycol is toxic; therefore, it should be handled under a chemical hood.

Antigen retrieval citrate buffer.

• •Sodium citrate buffer 10 mM: add 2.94 gr of Tri-sodium citrate dehydrated in 1 L ddH₂O and use a stirrer to dissolve it properly.
• •Add 0.5 mL of Tween-20 (final concentration 0.05%).

Store the solution at room temperature for maximum 3 months or at 4°C for longer storage.

Blocking solution

Reagent	Final concentration	Amount
Donkey serum	10%	500 μL
Triton X-100	0.3%	15 μL
PBS 0.1 M		4485 μL
Total		5 mL

Store at +4° for all the duration of the protocol.

Alternatives: Donkey serum can be replaced by other serums (e.g., fetal bovine serum, goat serum). If you use different types of serum, it is recommended that the host-species of the secondary antibodies is the same as the serum.

Sudan black solution.

• •Stock solution: Dissolve the Sudan Black powder in ethanol 70% for a final concentration of 1%.
• •Working solution: Dilute the stock solution in ethanol 70% to a final concentration of 0.1%. Filter the solution in order to eliminate the undissolved powder.

Store at room temperature, covered by light.

CRITICAL: Sudan Black under form of powder could be toxic if inhaled. Use a chemical hood to prepare the solution.

Step-by-step method details

This protocol is performed in a 6-multiwell plate (Note: the volume to add per well is at least 1 mL). The transfer of the tissue from one well to another is performed with a glass Pasteur pipette with a modified tip (i.e., hook-shaped) (troubleshooting 5). A brush can also be used, but it is not recommended as it can often tear/damage the tissue. All the incubation steps below (if not differently noted) are performed on a shaker (80 rpm).

Antigen retrieval

Timing: 15–30 min

Tissue fixation could mask the protein epitopes to which the primary antibody should bind. Antigen retrieval is highly recommended for the success of the protocol, especially when monoclonal antibodies are used. A change in pH and temperature will lead to the unmasking of the target. Here, we suggest a mild buffer (pH6), and a not-too-high temperature (no more than 100°C). Such parameters have been tested to prevent tissue damages and to allow antigen retrieval (for a wide range of specimens).

• 1.Wash the tissue section for few seconds in PBS 1× (to wash it from the anti-freezing solution and to acclimatize it to room temperature).
• 2.Heat the antigen retrieval buffer in a beaker until boiling (with a microwave or a laboratory heating stove). Insert the slice and keep the buffer boiling for 5 min (troubleshooting 4).

CRITICAL: Before using the section, let the antigen retrieval buffer cool down for around 30 min at room temperature to avoid a thermal shock.

• 3.Incubate the slice in PBS 1× (to recover the physiological pH).

Note: Processing multiple sections in one immunostaining session is possible (and recommended when comparing multiple specimens). However, it is recommended to process one section at a time for the antigen retrieval. Once you have finished the antigen retrieval for all sections, you can proceed to the next step and treat them in parallel.

Reduction of autofluorescence

Timing: 40 min

Timing: 30 min (for step 4)

Timing: 5 s (for step 5a)

Timing: 5 s (for step 5b)

Timing: 4 min (for step 5c)

One of the main issues of immunofluorescence on non-perfused or hyper-fixed tissues is the high autofluorescence background, often masking the antibody signal. Incubation in Sudan black allows a significant reduction of the background noise, leading to an increased signal/noise ratio.

• 4.Incubate the section with Sudan Black buffer (see materials and equipment).
• 5.
  Wash the slice.

  CRITICAL: Sudan Black buffer leaves a strong dark coloration in the well: it is better to transfer the tissue to a new multiwell.

  • a.
    Ethanol 70%.
  • b.
    Ethanol 50%.
  • c.
    PBS 1× (see troubleshooting 5).

Permeabilization and blocking

Timing: 1 h

Timing: 1 h (for step 6)

Incubation with blocking solution will permeabilize the cells (allowing a better penetration of the antibodies) and lower the non-specific binding of the antibodies.

Note: "If the antibody's target is localized on the membrane/transmembrane, you can avoid the use of triton in the blocking solution.

• 6.Incubate the slice with blocking solution (see materials and equipment) at room temperature, on the shaker.

Primary antibody labeling

Timing: overnight

In this step, the primary antibody will bind its target.

Note: Each antibody has its own dilution rate that should be experimentally determined. It is possible to use more than one antibody at the time in the same solution, as long as they are derived from different host-species.

• 7.Dilute the blocking solution 1:5 in PBS and add the antibody according to the right dilution (see key resources table).
• 8.Incubate overnight on a shaker at 4°C.

Pause point: If necessary, you can incubate the primary antibody for more than one night, up to 3. However, this step is not recommended, because long incubation times on the shaker can damage the tissue.

Secondary antibody labeling

Timing: 3 h

Timing: 10 min each wash (for step 9)

Timing: 2 h (for step 11)

Timing: 10 min each wash (for step 12)

Timing: 1 h (for step 13a)

Timing: 10 min each (for step 13a i)

Timing: 20 min (for step 13a ii)

Timing: 10 min each wash (for step 13a iii)

Timing: 1 day (for step 13b)

Timing: 10 min each (for step 13b i)

Timing: 10 min (for step 13b ii)

Timing: 1 h (for step 13b iii)

Timing: 10 min (for step 13b iv)

Timing: 5 min each (for step 13b v)

Timing: overnight (for step 13b vi)

The sample is now ready to be incubated with the proper secondary antibody conjugated with a fluorophore, for fluorescence detection.

Note: The dilution of the secondary antibody should be experimentally determined, however it is common to use a dilution rate 1:400–1:1000. To choose the right secondary antibody, see troubleshooting 6).

• 9.Wash the sample in PBS 1× for three times.
• 10.Dilute blocking solution 1:5 in PBS and add the right volume of secondary antibody according to the right dilution (see key resources table).
• 11.Incubate on the shaker at room temperature.

CRITICAL: secondary antibodies are light-sensitive, therefore from this step on, it is important to protect the multiwell plate with aluminum foil.

• 12.Wash the sample in PBS 1× for two times.

Nuclear staining

Nuclear staining is usually performed using 4,6-diamidino-2-phenylindole (DAPI) or Hoechst. However, these classical intercalants could not work on hyper-fixed tissues (e.g., tissues obtained from brain banks have been usually sitting in fixatives for months or years). A valid substitute is the NeuroTrace (the fluorescent counterpart of Nissl staining, (see key resources table). Here, we report both protocols, choose the proper one according to the fixation duration of the tissue. We recommend nuclear staining dilutions, but they need to be experimentally determined depending on the tissue.

• 13.
  Nuclear staining.

  • a.
    Incubate with DAPI/Hoechst at the dilution of 1:500 in PBS 1×.

    • i.
      Wash the sample with PBS 1× for two times on a shaker.
    • ii.
      Dilute DAPI or Hoechst in PBS 1× and incubate with the sample.
    • iii.
      Wash the sample in PBS for three times on a shaker.
  • b.
    Incubate with NeuroTrace at the dilution of 1:20 in PBS 1×.

    • i.
      Wash the sample in PBS 1× for two times on a shaker.
    • ii.
      Wash the sample in PBS 1× – 0.3% triton on a shaker
    • iii.
      Dilute the NeuroTrace in PBS 1× and incubate on the shaker at room temperature.
    • iv.
      Wash the sample in PBS 1× – 0.1% triton on a shaker.
    • v.
      Wash twice in PBS 1× on a shaker.
    • vi.
      Incubate in abundant PBS 1× at 4°C.

CRITICAL: To avoid damaging the tissue, it is suggested to avoid the shaker at this step.

Mounting and preservation

Timing: 1 day

• 14.Mount carefully the section on a glass-slide using a brush to prevent formation of folding and/or bubbles (troubleshooting 7).
• 15.Seal the slide using a mounting medium (we used Permount, see materials and equipment, but also other mounting medium can be used, as Mowiol) and let it dry overnight at room temperature.

CRITICAL: No shaker here.

• 16.Store the slide in a dark box at +4°C, to avoid signal bleaching.
• 17.Use a confocal microscope to image the samples for the best signal-to-noise ratio.

Expected outcomes

This protocol has been tested on brain tissue (specifically pre-frontal and frontal cortical regions) of a wide variety of species belonging to different mammalian orders: Rodentia, Primates, Chiroptera, Artiodactyla, and Carnivora (see key resources table and Figure 1A). The outcome is represented by an immunofluorescence staining with high signal/noise ratio, despite the challenging conditions of the tissues (e.g., tissue from non-model organisms; non-perfused and/or hyper-fixed tissue). This immunostaining protocol can be used to perform a single immunostaining (Figure 1), a double/triple immunostaining (Figure 2).

Figure 1.

Examples of outcome for single immunostainings performed on a wide range of species

The same immunostaining was first tested on a wide variety of species.

(A) On the left, evolutionary tree of the species tested with this protocol. This tree was created with https://phylot.biobyte.de, species taxonomy codes were obtained from NCBI taxonomy database. Scientific names correspond to the following common names: Mus musculus (mouse), Macaca mulatta (Rhesus macaque), Macaca fascicularis (crab-eating macaque), Homo sapiens (human), Pan troglodytes (chimpanzee), Carollia perspicillata (Seba’s short-tailed bat), Bos taurus (cattle), Tursiops truncatus (common bottlenose dolphin), Panthera leo (lion), Panthera tigris (tiger), Panthera pardus (leopard). On the right, indication of the correspondent orders. Color coded is as follows: Rodentia in gray; Primates in dark yellow; Chiroptera in purple; Artiodactyla in blue; Carnivora in orange.

(B–J) In green, immunostaining for Glial fibrillary acidic protein (GFAP) labeling astrocyte intermediate filament-III; in blue, NeuroTrace labeling nuclei. Species of the brain sections are indicated per each micrograph. Each micrograph is color labeled depending on the order the species belongs to. Scale bars = 20 μm.

Figure 2.

Examples of outcome for double immunostaining tested on a wide range of species

Different examples of double immunostaining on prefrontal cortex specimens from a selected species (i.e., cattle: Bos taurus). The same double immunostaining has been performed on the same species as in Figure 1A, not showed.

(A–A″) NeuroTrace (NT) in blue; S100b (a calcium binding protein expressed in an astrocyte subpopulation) in green; GFAP in red.

(B–B″) NT in blue, Aldh1l1 (a pan-astrocytic marker) in green; GFAP in red. Scale bars = 20 μm.

(C–C″) NT in blue, CD31 (a marker for endothelial cells) in green; GFAP in red. Full arrowheads point to double-stained cells; empty arrowheads point to single-stained cells. Scale bars = 20 μm.

Tissues shown in Figures are of 40 μm of thickness and the pictures have been taken with Nikon A1/R confocal, with a 60× oil objective. In order to include all the cells across the whole tissue thickness, images were taken as z-stacks of at least 42 steps of 1 μm, to include an optical section thickness of at least 40 μm. Images were subsequently processed as Z-stack MAX-projection modality in FIJI software (ImageJ).

Limitations

This protocol worked properly with all the antibodies we have tested (see key resources table). However, the proper antibody dilution should be determined according to the species, the thickness, and the fixation state of the sample. The protocol was tested only on mammalian species (see key resources table); but looks promising also for other vertebrates other than mammals, with similar tissue conditions.

Troubleshooting

Problem 1

Is it important to incubate in sucrose for at least two days and not just overnight?

Potential solution

Most of the tested samples needed at least two full days of incubation in sucrose, otherwise they could be damaged by low temperatures.

Problem 2

Is this protocol applicable only to free-floating sections?

Potential solution

The protocol is also applicable to tissues attached on glass slides. However, we have experienced that free-floating sections give better outcomes.

Problem 3

Is it necessary to cut at 40 μm?

Potential solution

The protocol is applicable to any type of thickness, but we found 40 μm is the best balance in order to have a good signal without damaging the sample.

Problem 4

While performing antigen retrieval, should the tissue boil or just be kept at a high temperature over 70°C?

Potential solution

We experienced a deterioration of fragile tissues if boiled. If the temperature is over 65°C–70°C, it is warm enough for melting the remaining paraffin.

Problem 5

Each time a new solution is used, is it better to replace the buffer in the same well or to move the tissue to a new one?

Potential solution

To avoid any damage to the tissue, it is better to replace the old solution with the new one in the same well, without moving the sample. However, in certain steps it is pivotal for the tissue to have a new and cleaned environment. These steps are highlighted as critical in the protocol.

Problem 6

How to choose a secondary antibody?

Potential solution

Secondary antibodies should be chosen according to: the host species of the primary antibody (e.g., α-mouse, if the primary antibody was produced in mouse); the color of the fluorophore (that has to match with the filters and the lasers of the microscope that is going to be used for the acquisitions), the serum used in the blocking solution (e.g., use secondary antibodies raised in donkey if you use a donkey serum).

Problem 7

How to mount the slices on a glass-slide?

Potential solution

We suggest immersing a glass slide in a petri-dish full of PBS 1×, moving the slice in the petri-dish and unfolding it carefully with a brush. Once the slice is in a correct position, start to suck out the PBS, letting the section lean on the slide.

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. Carmen Falcone (cfalcone@sissa.it).

Materials availability

This study did not generate new unique reagents.

Data and code availability

This study did not generate any unique datasets or code.