ALKALINE PHOSPHATASE PROCEDURE

Cut paraffin sections at 5 and place on APES treated slides (Superfrost + or Surgipath X-tra).

Heat immobilize sections in a 80C for 20 min.

Deparaffinize in 3 changes of xylene for 5 min each.

Hydrate in series of graded ethanols starting with 2 changes of absolute, one 95%, and ending with one 70%, all for 5 min each.

Rinse slides in distilled water for 2 min.

Rinse in 3 changes of PBS for 5 min each.

Apply blocking serum (monoclonals- Normal Rabbit serum 1:10) (Polyclonals-Normal Donkey Serum 1:5) for 20 min at room temp.

Drain excess serum and apply primary antibody for 60 min at room temp.

Rinse in 3 changes of PBS for 5 min each.

Apply biotinylatedsecondary antibody (Monoclonals: Rabbit-Mouse 1:200) (Polyclonals: Donkey-Rabbit Fab’2 1:800 {Jackson Immuno}). Incubate for 30 min at room temp.

Rinse in 3 changes of PBS for 5 min each.

Apply conjugated alkaline phosphatase - Streptavidin 1:50 {Biogenex} in PBS. Incubate for 30 min at room temp.

Rinse slides in 3 changes of PBS for 5 min each.

Apply fast red chromogen {Biogenex} (1 tablet + 5ml naphthol buffer). Incubate for 10 min at room temp.

Rinse slides in 2 changes of distilled water.

Counterstain with dilute Harris hematoxylin 1:10 for 2 min.

Rinse slides until clear in distilled water.

Immerse slides in Scotts water for 1 min.

Rinse slides in 2 changes of distilled water.

Mount with crystal mount or other aqueous mounting medium.

FAST RED IS AN ALCOHOL SOLUBLE CHROMOGEN!

DO NOT DEHYDRATE ALCOHOLS OR CLEAR IN XYLENE!