Introduction

The protocol below is written for using either purified or biotinylated primary antibodies for immunohistochemical staining of mouse frozen tissue sections. If one of our antibodies is applicable in immunohistochemistry based on published reports or feedback from our customers, we include this application on our datasheets as a reported usage. At eBioscience, only certain biotinylated antibodies against mouse cell surface antigens are tested following this protocol for immunohistochemical staining of acetone-fixed frozen tissue sections using peroxidase detection system. We do not test these antibodies on paraffin-embedded sections. Please note that most immunohistochemical staining of mouse tissues reported in the literature relates to frozen sections, rather than paraffin-embedded sections. If it is known that an antibody can be used on paraffin-embedded tissues, we include this information on the datasheets.

Useful Links

• IHC World Protocol Database (http://www.ihcworld.com) - This free access database contains nearly 300 protocols in the fields of histology, immunohistochemistry, antigen retrieval, antibody staining, electron microscopy and more.

Protocol for IHC Staining of Frozen Tissue Sections

Materials

• Tris-Saline: 0.05M Tris, 0.15M NaCl, pH 7.4 (Make 10X and filter, make 1000ml of 1X on the day of staining)
• Primary antibodies: purified or biotinylated formats
• Secondary antibodies for purified primary antibodies, depending on the species of the primary Ab:
  • Biotin-anti-Armenian Hamster IgG (Cat. No. 13-4113)
  • Biotin anti-Syrian Hamster IgG (Cat. No. 13-4213)
  • Biotin anti-rat IgG (Cat. No. 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, Cat. No. 195505)
• O.C.T. Compound (Sakura Finetek, Cat. No. 4583)
• Cryomold (Miles, Cat. No. 4557)
• Hematoxylin (optional)
• Acetone, Reagent grade
• Hydrogen peroxide (Sigma, Cat. No. H-1009)
• Mounting solution

Method

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. Then, put block on top of dry ice. Frozen blocks may be stored at -80°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°C.
3. Allow frozen blocks to equilibrate in the cryostat chamber for about 5 minutes. Cut 6-10mm sections. The best sections are usually obtained when the block temperature is around minus 18 to minus 20°C.
4. Let the sections dry for at least 30 minutes at room temperature. Note: Upon drying, tissues can be stored at 4°C for a few days; for longer-term storage up to a few months, store at -80°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 a PAP Pen and let it dry for a few minutes.
6. Add primary antibodies (diluted in 0.05M Tris-Saline, pH 7.4, 2.5% serum) directly onto the sections. Add in big droplets to cover the entire tissue at once to 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.
   1. If using biotinylated primary antibodies, skip the following 2 steps (8 and 9) and move to step 10.
   2. 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 100µl of diluted SA-AP or SA-HRP. Incubate for 30 minutes at room temperature.
11. Wash as described in step 7.
12. Stain slides.
    1. For HRP, incubate with AEC substrate: 25µl stock AEC (4mg/ml in DMSO) per 1ml 0.17 M NaOAc, pH 5.2 plus 1µl H₂0₂. Incubate for 20-30 minutes.
    2. For AP, incubate with AP substrate: Fast Violet (1mg/ml final) + Napthol AS-MX phosphate (0.2mg/ml final, from 10mg/ml stock in DMSO) in Tris-Saline pH 8.5 for 10-20 minutes until the desired positive staining is achieved.
13. Wash 2 times 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.