Immunohistochemistry Enzyme HRP

Staining Protocol




 1. Preparation of Slides


 A. Cell Lines

  • Grow cultured cells on sterile glass cover slips or slides overnight at 37 º C

  • Wash briefly with PBS
  • Fix as desired. Possible procedures include:

    10 minutes with 10% formalin in PBS (keep wet)

    5 minutes with ice cold methanol, allow to air dry

    5 minutes with ice cold acetone, allow to air dry

  • Wash in PBS

 B. Frozen Sections


  • Snap frozen fresh tissues in liquid nitrogen or isopentane pre-cooled in liquid nitrogen, embedded in OCT compound in cryomolds. Store frozen blocks at - 80 ºC.

  • Cut 4-8 um thick cryostat sections and mount on superfrost plus slides or gelatin coated slides. Store slides at - 80 ºC until needed.
  • Before staining, warm slides at room temperature for 30 minutes and fix in ice cold acetone for 5 minutes. Air dry for 30 minutes.
  • Wash in PBS

 C. Paraffin Sections

  • Deparaffinize sections in xylene, 2x5min.

  • Hydrate with 100% ethanol, 2x3min.
  • Hydrate with 95% ethanol, 1min.
  • Rinse in distilled water.
  • Follow procedure for pretreatment as required.

2. Pretreatments of Tissue Sections


Antigenic determinants masked by formalin-fixation and paraffin-embedding often may be exposed by epitope umasking, enzymatic digestion or saponin, etc. Do not use this pretreatment with frozen sections or cultured cells that are not paraffin-embedded.


3. Procedure


1.   Rinse sections in PBS-Tween 20 for 2x2min

2.   Serum Blocking: incubate sections with normal serum block – species same as secondary antibody, for 30 minutes to block non-specific binding of immunoglobulin. 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 block should be done after normal serum block.

3.   Primary Antibody: incubate sections with primary antibody at appropriate dilution in primary antibody dilution buffer for 1 hour at room temperature or overnight at 4 °C. Rinse in PBS-Tween 20

4.   Peroxidase Blocking: incubate sections in peroxidase blocking solution for 10 minutes at room temperature. Rinse in PBS-Tween 20.

5.   Secondary Antibody: incubate sections with biotinylated secondary antibody at appropriate dilution in PBS for 30 minutes at room temperature.

6.   Rinse in PBS-Tween 20 for 3x2min.

7.   Detection: incubate sections in streptavidin-HRP in PBS for 30 minutes at room temperature.

8.   Rinse in TBS for 3x2min.

9.   Chromogen/Substrate: incubate sections in DAB solution for 1-3 minutes.

10. Rinse in PBS-Tween 20 2x2min.

11. Counterstain if desire.

12. Rinse in distilled water.

13. Dehydrate through 95% ethanol for 2 min, 100% ethanol for 2x3min.

14. Clear in xylene for 2x5min.

15. Coverslip with mounting medium.