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Immunohistochemistry (IHC) Protocols: Indirect and Direct Methods

Detailed protocols to take you through tissue processing and on to direct and indirect methods of IHC labeling of floating sections.

Immunohistochemistry (IHC) allows you to detect a specific protein in tissue sections. In our experience, the optimal type of section is from frozen tissue fixed by transcardial perfusion with a buffered paraformaldehyde solution. This tissue will have been cut frozen in a cryostat and sections collected either by thaw mounting on slides (10–12 mm thick sections) or floating (30–36 mm thick sections).

The following protocols describe staining procedures of floating sections. Typically, floating sections are from tissues like adult brain and so require a thin brush to transfer them from one well to the next in a multi-well plate.

With indirect IHC methods, the primary antibody binds your protein of interest, and a secondary antibody conjugated to a reporter binds the primary antibody. There are more steps with the indirect method, but it is benefits from signal amplification since multiple secondary antibodies can bind the primary antibody. On the other hand, the indirect IHC methods can also produce more background than direct IHC methods in some situations.

For direct IHC, you use a primary antibody conjugated directly to a reporter, like an ATTO fluorophore. Since the direct IHC method doesn’t require a secondary antibody, it is quicker, cheaper, and may reduce non-specific binding. However, you may get a weaker signal compared with indirect IHC, especially if the protein of interest is present in low amounts.

Here we describe our IHC protocols that use rabbit-raised polyclonal primary antibodies on rat floating tissue sections.

Sacrifice and Tissue Processing

  1. Anesthetize the rats with pentobarbital sodium (Pental).
  2. Perform the transcardial perfusion, first with 50 ml of IHC phosphate-buffered saline (IHC-PBS) (Na2HPO4 0.016 M, KH2PO4 0.003 M, and NaCl 0.14 M) (pH 7.4), then with 220 ml of the fixative (ice-cold IHC-PBS with 4% paraformaldehyde (PFA) and 4% sucrose).
  3. Divide the tissue into coronal blocks and further fix by immersion in the fixative described above.
  4. Incubate at 4–8ºC overnight.
  5. Transfer the tissue blocks to 15% sucrose in IHC-PBS.
  6. Cut the tissue in a cryostat within 21 days.
  7. Float the tissue sections, 30 µm thick, in Cryopreservation Buffer (40% ethylene glycol, 1% polyvinylpyrrolidone, and 0.1 M potassium acetate buffer) (pH 6.5) and preserve at -20°C.

Proceed to EITHER IHC Protocols for Direct OR Indirect Labeling.

IHC Protocols for Indirect Labeling

To detect indirect IHC signals, use a bright-field or fluorescent microscope. It’s important to know which type of microscope you plan to use before beginning the experiment.

OPTION 1: Detection by Bright-Field Microscopy
OPTION 2: Detection by Fluorescent Microscopy

OPTION 1: Detection by Bright-Field Microscopy

  1. Rinse the floating sections wit IHC-PBS for 2 x 5 minutes.
  2. Quench endogenous peroxidase activity by incubating the sections with 0.2% hydrogen peroxide in IHC-PBS 0.2% Triton X-100*, and 20% methanol for 25 minutes at room temperature.
  3. Rinse the sections with IHC-PBS for 2 x 5 minutes.
  4. If antigen retrieval is necessary, proceed to the protocol on enzymatic retrieval in the troubleshooting section at the end of this protocol. Please note that treatment with hydrogen peroxide (step 2) induces moderate antigen retrieval.
  5. Rinse the sections with IHC-PBS for 2 x 5 minutes.
  6. Incubate the sections with the rabbit primary antibody in a solution containing IHC-PBS, 0.3% Triton X-100*, 0.05% Tween-20, and 2% normal serum (NS)**, for 1 hour at room temperature.
  7. Transfer to 4°C overnight.
  8. Rinse the sections with IHC-PBS containing 2% NS** for 2 x 5 minutes.

*If your primary antibody targets an extracellular protein, reduce the Triton X-100 to 0.05% in both the primary and secondary antibody solutions.
**Use a serum based on the species that your secondary antibody was raised in. For example, if your secondary antibody was raised in donkeys, use normal donkey serum (NDS). Likewise, if your secondary antibody was raised in goats, use normal goat serum (NGS).

Two options for indirect bright-field IHC with secondary antibodies are available here:
1A: Secondary antibody conjugated to biotin
1B: Secondary antibody conjugated to HRP

OPTION 1A: Secondary Antibody Conjugated to Biotin

  1. Incubate the sections with biotinylated donkey anti-rabbit antibody (Merck, catalog no. AP182B) diluted 1:400 in IHC-PBS, containing 0.3% Triton X-100*, 0.05% Tween-20, and 2% NDS, for 1 hour at room temperature.
  2. Transfer to 4°C overnight.
  3. Rinse the sections with IHC-PBS containing 2% NDS** for 2 x 5 minutes.
  4. Incubate the sections with extravidin-peroxidase (Merck, catalog no. E2886) diluted 1:200 in IHC-PBS, for 1 hour at room temperature.
  5. Proceed to Detection with a Bright-Field Microscope.

*If your primary antibody targets an extracellular protein, reduce the Triton X-100 to 0.05% in both the primary and secondary antibody solutions.
**Use a serum based on the species that your secondary antibody was raised in. For example, if your secondary antibody was raised in donkeys, use NDS. Likewise, if your secondary antibody was raised in goats, use NGS.

OPTION 1B: Secondary Antibody Conjugated to HRP

  1. Incubate the sections with horseradish peroxidase (HRP)-conjugated donkey anti-rabbit antibody (Merck, catalog no. AP182P) diluted 1:400 in IHC-PBS, 0.3% Triton X-100*, 0.05% Tween-20, and 4% NDS, for 1 hour at room temperature.
  2. Transfer to 4°C overnight.
  3. Rinse the sections with IHC-PBS containing 2% NDS** for 2 x 5 minutes.
  4. Proceed to Detection with a Bright-Field Microscope.

*If your primary antibody targets an extracellular protein, reduce the Triton X-100 to 0.05% in both the primary and secondary antibody solutions.
**Use a serum based on the species that your secondary antibody was raised in. For example, if your secondary antibody was raised in donkeys, use NDS. Likewise, if your secondary antibody was raised in goats, use NGS.

Detection with a Bright-Field Microscope

  1. Incubate the sections with a solution containing 0.0125% diaminobenzidine (DAB; Merck, catalog no. D5637) and 0.05% nickel ammonium sulfate for 10 minutes at room temperature.
  2. Transfer the sections to the same DAB solution described above, which has been supplemented with hydrogen peroxide at a final concentration of 0.0015%. This is necessary to monitor the color reaction.
  3. Rinse the sections with IHC-PBS for 4 x 10 minutes.
  4. Mount the sections on glass slides (gelatinized or coated with any other type of adhesive material) and allow them to dry.
  5. Dehydrate the sections by incubation with increasing ethanol concentrations (70%, 90%, and 100%; 5 minutes at each concertation). Delipidate in xylene for 10 minutes and apply a coverslip with Permount (or any other xylene diluted adhesive).
  6. Detect with a bright-field microscope.

Example Data

Expression of IP3 receptor 1 in mouse cerebellum. Immunohistochemical staining of mouse cerebellum with Anti-IP3 Receptor-1 (ITPR1) Antibody (ACC-019). A) Immunoreactivity appeared in Purkinje cells (indicated with arrows) and their dendritic trees. B) Axonal processes coursing through cerebellar white matter were visualized (shown by the arrow).

OPTION 2: Detection by Fluorescent Microscopy

  1. Rinse the floating sections with IHC-PBS for 2 x 5 minutes.
  2. If antigen retrieval is necessary, proceed to the protocol on enzymatic retrieval in the troubleshooting section at the end of this protocol.
  3. Rinse the sections with IHC-PBS for 2 x 5 minutes.
  4. Incubate the sections with the rabbit primary antibody in a solution containing IHC-PBS, 0.3% Triton X-100*, 0.05% Tween-20, and 2% NS** for 1 hour at room temperature.
  5. Transfer to 4°C overnight.
  6. Rinse the sections with IHC-PBS containing 2% NDS for 2 x 5 minutes.

*If your primary antibody targets an extracellular protein, reduce the Triton X-100 to 0.05% in both the primary and secondary antibody solutions.
**Use a serum based on the species that your secondary antibody was raised in. For example, if your secondary antibody was raised in donkeys, use NDS. Likewise, if your secondary antibody was raised in goats, use NGS.

Two options for indirect fluorescent IHC with secondary antibodies are available here:
2A: Secondary antibody conjugated to biotin
2B: Secondary antibody conjugated to a fluorophore

OPTION 2A: Secondary Antibody Conjugated to Biotin

  1. Incubate the sections with biotinylated donkey anti-rabbit antibody (Merck, catalog no. AP182B) diluted 1:400 in IHC-PBS, 0.3% Triton X-100*, 0.05% Tween-20, and 2% NDS**, for 1 hour at room temperature.
  2. Transfer to 4°C overnight.
  3. Rinse the sections with IHC-PBS containing 2% NDS for 2 x 5 minutes.
  4. Incubate the sections with streptavidin-Cy3 (Sigma, catalog no. S6402) diluted 1:200 in IHC-PBS, for 1 hour at room temperature, protected from light.
  5. Proceed to Detection with a Fluorescent Microscope.

* If your primary antibody targets an extracellular protein, reduce the Triton X-100 to 0.05% in both the primary and secondary antibody solutions.
** Use a serum based on the species that your secondary antibody was raised in. For example, if your secondary antibody was raised in donkeys, use NDS. Likewise, if your secondary antibody was raised in goats, use NGS.

OPTION 2B: Secondary Antibody Conjugated to a Fluorophore

  1. Incubate the sections with a fluorophore-conjugated goat anti-rabbit antibody diluted 1:200 in IHC-PBS, 0.3% Triton X-100*, 0.05% Tween-20, and 2% NGS**, for 1 hour at room temperature.
  2. Transfer to 4°C overnight.
  3. Rinse the sections with IHC-PBS containing 2% NGS** for 2 x 5 minutes.
  4. Proceed to Detection with a Fluorescent Microscope.

* If your primary antibody targets an extracellular protein, reduce the Triton X-100 to 0.05% in both the primary and secondary antibody solutions.
** Use a serum based on the species that your secondary antibody was raised in. For example, if your secondary antibody was raised in donkeys, use NDS. Likewise, if your secondary antibody was raised in goats, use NGS.

Detection with a Fluorescent Microscope

  1. Mount the sections on glass slides in IHC-PBS (pH 7.4).
  2. Dry the glass slides in a fume hood for 1 hour.
  3. Stain the sections on the slides with DAPI by placing DAPI solution (5 mg/ml stock solution in deionized water or dimethylformamide (DMF); dilute to 500 nM in IHC-PBS for final use) on each slide. Next, cover each slide with a piece of parafilm to spread the solution evenly on each slide.
  4. After 2 minutes, remove the parafilm and rinse the slide with 0.5 ml IHC-PBS using a pipette (repeat twice).
  5. Dry the slides in a fume hood for 30 minutes.
  6. Apply coverslips using the adhesive Immu-MountTM (ShandonTM).
  7. Dry the slides overnight, protected from light.
  8. Store at -18°C until they ready to view under the microscope.

Example Data

Glucose transporter 3 (GLUT3) expression in the murine hippocampus and cerebellum. Immunohistochemical staining of perfusion-fixed frozen mouse brain sections with Anti-GLUT3 (extracellular) Antibody (AGT-023) (1:200) and goat anti-rabbit Alexa Fluor 488. A) GLUT3 staining (green) in the mouse hippocampal dentate gyrus is detected in interneurons (arrows pointing up) in the hilus and granule layer arrow pointing down). The cell nuclei are stained with DAPI (blue). B) GLUT3 staining (green) in the mouse cerebellum is observed in Purkinje cells (vertical arrows) and dendrites (horizontal arrows) in the molecular layer (Mol). The cell nuclei are stained with DAPI (blue).

P2RY6 expression in the rat parietal cortex. Immunohistochemical staining of perfusion-fixed frozen rat brain sections with Anti-P2Y6 Receptor (extracellular) Antibody (APR-106) (1:1,000), donkey anti-rabbit biotin-conjugated antibody, and streptavidin-Cy3. P2RY6 immunoreactivity (red) appeared in the pyramidal neurons (arrows). The cell nuclei are stained with DAPI (blue).

IHC Protocol for Direct Labeling

  1. Rinse the floating sections in IHC-PBS for 2 x 5 minutes.
  2. If antigen retrieval is necessary, please consult the troubleshooting section at the end of this protocol.
  3. Rinse the sections in IHC-PBS for 2 x 5 minutes.
  4. Incubate the sections with the primary antibody diluted in a solution containing IHC- PBS, 0.05% Triton X-100, 0.05% Tween-20, and 2% NS**, for 1 hour at room temperature.
    Note: When using ATTO-labeled antibodies, the optimal dilution should be initially tested with a relatively low dilution (e.g., 1:60). The dilution should then be increased in order to optimize the signal-to-background ratio.
  5. Incubate the sections at 4–8ºC overnight.
  6. Rinse the sections in IHC-PBS, containing 2% NS** for 2 x 5 minutes.
  7. Proceed to Detection with a Fluorescent Microscope.

**Use a serum based on the species that your secondary antibody was raised in. For example, if your secondary antibody was raised in donkeys, use NDS. Likewise, if your secondary antibody was raised in goats, use NGS.

Detection with a Fluorescent Microscope

  1. Mount the sections on glass slides in IHC-PBS (pH 7.4).
  2. Dry the slides in a fume hood for 1 hour.
  3. Stain the sections on the slides with DAPI by placing DAPI solution (5 mg/ml stock solution in deionized water or dimethylformamide (DMF); dilute to 500 nM in IHC-PBS for final use) on each slide. Next, cover each slide with a piece of parafilm to spread the solution evenly on each slide.
  4. After 2 minutes, remove the parafilm and rinse the slide with 0.5 ml of distilled deionized water using a pipette (repeat twice).
  5. Dry the slides in a fume hood for 30 minutes.
  6. Apply coverslips using the adhesive Immu-MountTM (ShandonTM).
  7. Dry the slides overnight, protected from light.
  8. Store at -18°C until they are viewed under the microscope.

Example Data

CaVβ2 expression in the rat brain. Immunohistochemical staining of the rat hippocampus using Anti-CACNB2-ATTO Fluor-594 Antibody (ACC-105-AR). CaVβ2 staining (red) appeared in the CA3 pyramidal layer (P; arrows). The cell nuclei are stained with DAPI (blue).

5-Hydroxytryptamine receptor 1B (HTR1B) expression in rat and mouse brains. Immunohistochemical staining of rat and mouse brain sections using Anti-5HT1B Receptor (HTR1B) (extracellular)-ATTO Fluor-488 Antibody (ASR-022-AG) (1:80). A) 5-HT1B receptor staining (green) in the rat hippocampal CA1 region was detected near the pyramidal layer (P) and in apical dendrites (arrows). B) In the mouse hippocampal CA1 region, 5-HT1B receptor staining (green) appeared in the pyramidal layer (P) and in apical dendrites. The cell nuclei are stained with DAPI (blue).

Troubleshooting

Antigen Retrieval

If there is no observed staining after you IHC experiment, then we recommend that one of the following antigen retrieval protocols. You can use both methods, but you will need to determine the optimal conditions with careful testing.

Hydrogen peroxide treatment (moderate treatment)

  1. Incubate the sections with 0.2% hydrogen peroxide in IHC-PBS (pH 7.4), 0.2% Triton X-100*, and 20% methanol, for 25 minutes at room temperature.

*If your primary antibody targets an extracellular protein, reduce the Triton X-100 to 0.05%.

Enzymatic retrieval (aggressive treatment)

Stock solutions required for trypsin treatment:

  • Trypsin type II-S (Sigma, catalog no. T-8128): 0.1% trypsin dissolved in IHC-PBS. Store as frozen aliquots.
  • Trypsin type II-S inhibitor (Sigma, catalog no. T-9128): 0.1% trypsin inhibitor diluted in IHC-PBS. Store as frozen aliquots.
  1. Dilute the trypsin stock solution 1:100 to obtain a final trypsin concentration of 0.001%.
  2. Add CaCl2 to the trypsin stock solution for a final concentration of 0.001%.
  3. Incubate the sections in this solution for 5–7 minutes at 37°C.
  4. Rinse the sections with IHC-PBS for 2 x 5 minutes.
  5. Dilute the trypsin inhibitor stock solution 1:100 into the primary antibody solution*.

*It’s incredibly important to remember that if you use trypsin in this step, then you must also add 0.001% trypsin inhibitor to the primary antibody solution.

High Background with Biotin-Avidin Amplification (for OPTION 2A)

High background is likely due to endogenous avidin-binding sites in tissues. The biotin-conjugated secondary antibody will bind streptavidin or extravidin conjugated to peroxidase or a fluorophore. However, the streptavidin or extravidin may bind not only to the biotin on the secondary antibody, but also to additional binding sites in the tissue.

To overcome this problem, the endogenous avidin binding sites need to be blocked before adding the primary antibody. You can achieve this by incubating the tissue sections in a solution containing non-conjugated streptavidin.

To block non-specific avidin binding sites in tissues, we use a kit from Vector Laboratories (catalog no. SP-2002). If you require this treatment for high background levels, perform this step after antigen retrieval (if required) and after rinsing the slides for 2 x 5 minutes in IHC-PBS.

Pre-saturation of endogenous avidin binding ability

  1. Add 4 drops from the “streptavidin” bottle to 5 ml of IHC-PBS.
  2. Incubate the sections in this solution for 30 minutes at room temperature.
  3. Rinse the sections for 2 x 5 minutes in IHC-PBS.

Saturation of residual biotin binding potential

  1. Add 4 drops from the “biotin” bottle to 5 ml of IHC-PBS.
  2. Incubate the sections in this solution for 30 minutes at room temperature.
  3. Rinse the sections for 2 x 5 minutes in IHC-PBS.
  4. Start you IHC experiment by incubating the sections with the primary antibody.