Immunohistochemistry Protocol

Frozen Sections:

1. Snap frozen fresh tissues in liquid nitrogen or isopentane pre-cooled in liquid nitrogen, embedded in OCT compound in cryomolds. Store frozen blocks at -80C.
2. Cut 4-8 um thick cryostat sections and mount on superfrost plus slides or gelatin coated slides. Store slides at - 80C until needed.
3. Before staining, warm slides at room temperature for 30 minutes and fix in ice cold acetone for 10 minutes. Air dry for 30 minutes.
4. Wash in PBS.

Paraffin Sections:

1. Deparaffinize sections in xylene, 2x5min.
2. Hydrate with 100% ethanol, 2x3min.
3. Hydrate with 95% ethanol, 1min.
4. Rinse in distilled water.

Tissue Staining:

1. Follow procedure for pretreatment as required.
2. Procedure for Immunoenzyme Staining.
3. Rinse Sections in Washing Buffer (PBS+0.1% Triton X-100, pH 7.4) for 2x2 min.
4. Serum Blocking: incubate sections in normal serum block – species same as secondary antibody( Wash Buffer + 5% Normal Sera). Note: since this protocol uses avidin-biotin detection system, avidin/biotin block may be needed based on tissue type. If you do, the avidin/biotin blocking should be done after normal serum block and before primary antibody incubation.
5. Primary Antibody: incubate sections in primary antibody at appropriate dilution in Wash Buffer for 1 hour at room temperature or overnight at 4C. Note:Do not rinse sections between serum block and primary antibody incubation.
6. Rinse in washing buffer for 3x2 min.
7. Peroxidase Blocking: incubate sections in peroxidase blocking solution for 10 minutes at room temperature.
8. Rinse in washing buffer for 3x2 min.
9. Secondary Antibody: incubate sections in biotinylated secondary antibody at an appropriate dilution in Wash Buffer for 30 minutes at room temperature.
10. Rinse in washing buffer for 3x2 min.
11. Detection: incubate sections in streptavidin-HRP in Wash Buffer for 30 minutes at room temperature.
12. Rinse in washing buffer for 3x2 min.
13. Chromagen/Substrate: incubate sections in peroxidase substrate solution.
14. Rinse in washing buffer for 3x2 min.
15. Rinse in running tap water for 2-5 minutes.
16. Dehydrate through 95% ethanol for 1 minute, 100% ethanol for 2x3min.
17. Clear in xylene for 2x5min.
18. Coverslip with mounting medium.

 
Peroxidase Block:

    0.3% H2O2 in PBS (for Paraffin Sections)

    0.3% H2O2 in Methanol (for Frozen Sections)
 

Avidin/Biotin Block:

    0.001% Avidin in PBS

    0.001% Biotin in PBS

        Block for 10 minutes with first solution, rinse with PBS, block for 10 minutes with second solution.

 
*The above information is only intended as a guide. The researcher should determine what protocol best meets their needs. Please follow safe laboratory procedures.