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eBioscience: Immunohistochemistry (IHC) Staining, Frozen Tissues

Immunohistochemistry Staining, Frozen Tissues (Acetone-Fixed)

The protocol below is written for using either Purified or Biotinylated primary antibodies for immunohistochemical staining. At eBioscience, a selected number of Biotinylated antibodies against mouse cell surface antigens are tested for immunohistochemical staining of frozen sections using Peroxidase detection system.

What you need:

  • Tris-Saline: 0.05 M Tris, 0.15 M NaCl, pH 7.4 (Make 10X and filter, make 1000 ml of 1X on the day of staining)
  • Primary antibodies:
    • Purified or Biotinylated formats
  • Secondary antibodies: For Purified primary antibodies, the following Biotinylated secondary antibodies, depending on the species of the primary Ab:
    • Biotin-anti-Armenian Hamster IgG, 13-4113
    • Biotin anti-Syrian Hamster IgG, 13-4213
    • Biotin anti-rat IgG, 13-4813
  • Enzymes:
    • Streptavidin conjugated horseradish peroxidase (SA-HRP) or
    • Streptavidin conjugated alkaline phosphatase (SA-AP)
  • Substrates:
    • For HRP: AEC
    • For AP: Fast Violet
  • Blocking reagents: FBS or mouse serum
  • PAP-PEN (Research Products International, 195500)
  • O.C.T. Compound (Sakura Finetek, 4583)
  • Cryomold (Miles, 4557)
  • Hematoxylin (optional)
  • Acetone, Reagent grade
  • Hydrogen peroxide (Sigma, H-1009)
  • Mounting solution


1) Embed fresh tissues carefully in OCT in plastic mold, taking care not to trap air bubbles surrounding the tissue. Freeze tissue by setting mold on top of liquid nitrogen until 70-80% of the block turns white and then put block on top of dry ice. The frozen blocks may be stored at minus 80 degrees C for long-term storage.

2) For cutting step, mount the frozen block on the cryostat holder. Never at any point let the tissue warm up to temperatures above minus 15 degrees C.

3) Allow frozen blocks to equilibrate in the cryostat chamber for about 5 minutes. Cut 6-10 mm sections. The best sections are usually obtained when the block temperature is around minus 18 to minus 20 degrees C.

4) Let the sections dry for at least 30 minutes at room temperature.

Note: Upon drying, tissues can be stored at 4 degrees C for a few days; for longer-term storage up to a few months, place in minus 80 degrees C.

5) Fix the sections by immersing in acetone jar for 1-2 minutes at room temperature, and let air-dry. Carefully draw boundaries of sections using the PAP PEN and let it dry for a few minutes.

6) Add primary antibodies (diluted in 0.05 M Tris-Saline, pH 7.4, 2.5% serum) directly onto the sections. Add in big droplets to cover the entire tissue at once; this will prevent fracturing of the sections due to the surface tension of the solution. Incubate in a chamber for at least one hour at room temperature.

Note: Once the sections are re-hydrated with Tris-saline, never let the tissue dry out again (this will ruin the tissue architecture).

7) Wash the sections gently in Tris-saline for 3-5 minutes and then in Tris-saline/2.5% serum for another 3-5 minutes.

If using Biotinylated primary antibodies, skip the following 2 steps (8 and 9) and move to step 10.
If using Purified primary antibodies, continue the protocol at step 8.

8) Without letting the sections dry out, add the secondary antibody diluted in Tris-saline/2.5% serum. Incubate as before for at least 45 minutes.

9) Wash as described in step 7.

10) Cover slides with about 100ul of diluted SA-AP or SA-HRP. Incubate for 30 minutes at room temperature.

11) Wash as described in step 7.

12) For HRP, incubate with AEC substrate: 25ul stock AEC (4 mg/ml in DMSO) per 1 ml 0.17M NaOAc, pH 5.2 plus 1ul H202. Incubate for 20-30 minutes.

For AP, incubate with AP substrate: Fast Violet (1 mg/ml final) + Napthol AS-MX phosphate (0.2 mg/ml final, from 10 mg/ml stock in DMSO) in Tris-Saline pH 8.5 for 10-20 minutes until the desired positive staining is achieved.

13) Wash 2X in Tris-saline.

14) Optional step: Counter-stain with Mayer's hematoxylin for 30 seconds and wash with tap water for 2-5 minutes.

15) Mount coverslips with mounting media.

16) Read the slides.


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