HORSERADISH PEROXIDASE PROCEDURE

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

Heat immobilize sections in a 80 C 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.

Block endogenous peroxidase using 3% H2O2 for 5 min.

Rinse slides in several changes of distilled water.

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

Apply primary antibody for 60 min. or longer at room temp.

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

MOUSE PRIMARY MONOCLONALS ONLY - Apply Rabbit-Mouse biotinylated secondary antibody 1:200 {Dako} in 10% normal rabbit serum. Incubate for 30 min. at room temp.

RABBIT PRIMARY POLYCLONALS ONLY - Apply Donkey-Rabbit biotinylated secondary Fab'2 antibody 1:800 {Jackson} 10% normal Donkey serum. Incubate for 30 min. at room temp.

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

Apply conjugated Horseradish peroxidase - Streptavidin 1:1000 {Dako}. Incubate for 30 min. at room temp.

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

Apply DAB (1.5mg DAB + 2.5ml PBS + 10m l H2O2). Incubate for 10 - 30 min. at room temp.

Rinse in 2 changes of distilled water.

Hematoxylin Counterstain - Immerse in dilute (1:10) Harris hematoxylin for 2 min.

Rinse slides until clear in distilled water.

Immerse slides Scotts water for 1 min.

Rinse in 2 changes of distilled water.

Dehydrate in 95% ETOH (6 dips)

Dehydrate in 2 changes of absolute ETOH (10 dips each).

Clear in 3 changes of xylene.

Mount with permount or other synthetic mounting medium.