Beta Catenin Staining Protocol

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

Heat immobilize sections in an 80C for 20 min.

Deparaffinize the sections in xylene 3 X 5 min. each.

Place the sections in absolute ETOH 2 X 5 min. each.

Hydrate in 95% ETOH for 5 min.

Hydrate in 70% ETOH for 5 min.

Rinse in D.I. H2O for 2 min.

Remove carmine stain if necessary using 0.5% lithium carbonate for 5 min.

Rinse in D.I. H2O for 2 min if it was necessary to perform previous step

Perform microwave antigen retrieval  

Block endogenous peroxidase activity using 3% H2O2 for 5 min.

Rinse in dH2O for 2 min.

Rinse slides in PBS 3 X 5 min. each.

Apply anti b-catenin (1:25) and incubate for 1 hour.

Rinse slides in PBS 3 X 5 min. each.

Apply biotinylated Rabbit anti-Mouse (1:200 w/ 10% NRS) incubate for 30 min.

Rinse slides in PBS 3 X 5 min. each.

Apply conjugated HRP streptavidin (1:1000) incubate to 30 min.

Rinse slides in PBS 3 X 5 min. each.

Apply DAB (3 mg DAB in 5ml PBS + 20l of H2O2) incubate for 10 min.

Rinse slides in dH2O 3 X 1 min. each.

Counterstain using dilute (1:10) Harris hematoxylin for 2 min.

Dehydrate, clear and mount with permount.