General IHC Protocol

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

Frozen Sections

  1. Snap-freeze fresh tissues in liquid nitrogen or isopentane pre-cooled in liquid nitrogen, embedded in OCT compound in cryomolds. Store frozen blocks at -80º C.

  2. Cut 4-8 m m thick cryostat sections and mount on superfrost plus slides or gelatin coated slides. Store slides at - 80º C 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 (Rockland code # MB-008).

Paraffin Sections

  1. Deparaffinize sections in xylene, 2x5min.

  2. Hydrate with 100% ethanol, 2x3min.

  3. Hydrate with 95% ethanol, 1min.

  4. Rinse in ultra pure water (Rockland code # MB-009-1000).

Procedure for Immunoenzyme Staining

  1. Follow procedure for pretreatment as required.

  2. Rinse Sections in PBS for 2x2 min (Rockland code # MBS-008).

  3. Serum Blocking: incubate sections in normal serum block ? species same as secondary antibody (for example, Normal Goat Serum (NGS) Rockland code #B-304 if secondary antibody is goat host).
    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.

  4. Primary Antibody: incubate sections in primary antibody at appropriate dilution in dilution buffer (PBS supplemented with serum block) for 1 hour at room temperature or overnight. Note: Do not rinse sections between serum block and primary antibody incubation.

  5. Rinse in PBS buffer for 3x2 min.

  6. Peroxidase Blocking: incubate sections in 1% hydrogen peroxidase in PBS (Rockland code #KHJ001) for 10 minutes at room temperature.

  7. Rinse in PBS buffer for 3x2 min.

  8. Secondary Antibody: incubate sections in biotinylated secondary antibody (1:500, for example Rockland code # 611-106-122) in PBS buffer for 30 minutes at room temperature.

  9. Rinse in PBS buffer for 3x2 min.

  10. Detection: incubate sections in streptavidin-HRP (1:500, Rockland code # S000-03) in PBS buffer for 30 minutes at room temperature.

  11. Rinse in PBS buffer for 3x2 min.

  12. Chromagen/Substrate: incubate sections in peroxidase substrate solution. Use Rockland code #TMB-M or code #DAB-10 for stable blue or brown staining, respectively.

  13. Rinse in PBS buffer for 3x2 min.

  14. Rinse in ultra pure water for 3x5 min.

  15. Dehydrate through 95% ethanol for 1 minute, 100% ethanol for 2x3min.

  16. Clear in xylene for 2x5min.

  17. Coverslip with mounting medium (Rockland code # KHH001).

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