CD-31 (PECAM-1) Staining Protocol

Updated 06/19/2003

Cut formalin fixed paraffin sections at 4m and place on APES treated slides, i.e. (Superfrost + or Surgipath X-tra).

Heat immobilize sections in an 80C for 30 min.

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

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

Hydrate in 95% ETOH for 5 min.

Hydrate in 70% ETOH for 5 min.

Rinse in deionized (DI) H2O for 2 min.

Remove carmine stain if necessary using 0.5% lithium carbonate for 5 min. Note: this step is only necessary for mammary gland sections obtained from carmine stained whole mounts.

Rinse in DI H2O for 2 min if it was necessary to perform previous step

Perform  HIER in pressure cooker or use microwave antigen retrieval technique

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

Rinse in DI H2O for 2 min.

Rinse slides in Phophate Buffered Saline (PBS) 3 X 5 min. each.

Use a hydrophobic barrier pen (PAP pen) {Zymed cat. # 00-8888} to isolate tissue sections on each slide.

Apply Normal Donkey Serum (1:10) {Jackson Immuno Research cat. # 017-000-121} and incubate for 20 at room temp (RT).

Drain excess serum and apply Goat anti-CD-31 (1:750) {Santa Cruz Biotech Cat.# SC-1506}, incubate for 1 hour at RT.

Rinse slides in PBS 3 X 5 min. each.

Apply biotinylated Donkey anti-Goat IgG (1:500) {Jackson Immuno Research cat. # 705-065-147}, incubate for 30 min at RT.

Rinse slides in PBS 3 X 5 min. each.

Apply conjugated HRP streptavidin (1:1000) {Dako cat. # P397}, incubate to 30 min at RT.

Rinse slides in PBS 3 X 5 min. each.

Apply stable DAB {Research Genetics cat. # 750118} and incubate for 6 min at RT.

Rinse slides in DI H2O 3 X 1 min. each.

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

Rinse until clear, blue in Scott's water and rinse.

Dehydrate in 95 ETOH 2 x 2 min.

Dehydrate in absolute ETOH 2 x 2 min.

Clear in xylene, 3 x 2 min

Mount with permount or other synthetic resin and coverslip.