A detailed protocol to take you through tissue processing and on to the direct IHC method of IHC for floating sections using a fluorescent microscope.
Immunohistochemistry (IHC) allows you to detect a specific protein in tissue sections. We find the optimal section is from frozen tissue fixed by transcardial perfusion with a buffered paraformaldehyde solution. Sections are cut on cryostat at 10–12 µm thick sections for slides or 30–36 µm thick for floating sections.
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 when compared to an indirect IHC protocol. However, you may get a weaker signal than if you were using indirect IHC, especially if the protein of interest is present in low amounts.
Here we describe our IHC protocol that uses rabbit-raised polyclonal primary antibodies on rat floating tissue sections.
If you have any problems, please see our extensive troubleshooting guides.
Sacrifice and Tissue Processing
- Anesthetize the rats with pentobarbital sodium (Pental).
- Perform the transcardial perfusion, first with 50 ml/rat of IHC phosphate-buffered saline (IHC-PBS), then with 220 ml/rat of ice-cold Fixative Buffer.
|IHC-PBS (pH 7.4)|
|Na2HPO4||0.2 M||80 ml|
|NaH2PO4||0.2 M||16 ml|
|Double distilled water||860 ml|
|Fixative Buffer (pH 7.4)|
|Na2HPO4||0.2 M||120 ml|
|NaH2PO4||0.2 M||24 ml|
- Divide the tissue into coronal blocks and further fix by immersion in the fixative described above.
- Incubate at 4–8°C overnight.
- Transfer the tissue blocks to Perfusion Buffer.
|Perfusion Buffer (pH 7.4)|
|Na2HPO4||0.2 M||220 ml|
|NaH2PO4||0.2 M||50 ml|
|Double distilled water||270 ml|
- Cut the tissue in a cryostat within 21 days.
- Float the tissue sections, 30 µm thick, in Cryopreservation Buffer and preserve at -20°C.
|Cryopreservation Buffer (pH 6.5)|
|Potassium acetate buffer||0.1 M|
Protocol for Direct IHC
- Rinse the floating sections in IHC-PBS for 2 x 5 minutes.
- If antigen retrieval is necessary, please see antigen retrieval section at the end.
- Rinse the sections in IHC-PBS for 2 x 5 minutes.
- Incubate the sections with the primary antibody diluted in Direct Antibody Solution for 1 hour at room temperature.
|Direct Antibody Solution|
|Reagent||% of final volume|
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 to optimize the signal-to-background ratio.
- Incubate the sections at 4–8°C overnight.
- Rinse the sections in IHC-PBS, containing 2% NS for 2 x 5 minutes.
- Mount the sections on glass slides in IHC-PBS (pH 7.4).
- Dry the slides in a fume hood for 1 hour.
- 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.
- After 2 minutes, remove the parafilm and rinse the slide with 0.5 ml of distilled deionized water using a pipette (repeat twice).
- Dry the slides in a fume hood for 30 minutes.
- Apply coverslips using the adhesive Immu-MountTM (ShandonTM).
- Dry the slides overnight, protected from light.
- Store at -18°C until they are viewed under the microscope.
Antigen Retrieval Protocol
If there is no observed staining after your 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)
- 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.
- Dilute the trypsin stock solution 1:100 to obtain a final trypsin concentration of 0.001%.
- Add CaCl2 to the trypsin stock solution for a final concentration of 0.001%.
- Incubate the sections in this solution for 5–7 minutes at 37°C.
- Rinse the sections with IHC-PBS for 2 x 5 minutes.
- 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.
Figure 1. 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).
Figure 2. 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).