Immunohistochemistry Enzyme Alkaline Phosphatase (AP)

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 for Immunoenzyme (AP) Staining


1.   Rinse sections in TBS-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.

4.   Rinse in TBS-Tween 20 for 3x2min.

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

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

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

8.   Rinse in TBS for 3x2min.

9.   Chromogen/Substrate: incubate sections in AP substrate solution for 30 minutes to several hours.

10. Rinse in distilled water 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.