Biotinylated goat anti rabbit

The sections were de-paraffinized and hydrated as previously mentioned in the H&E and Masson’s trichrome staining protocols. Thereafter, these were pretreated in Target Retrieval Solution (Dako North America, Inc., CA, USA) and then washed in water for 5 min and rinsed in distilled water. Next, the sections were treated with 3% Hydrogen Peroxide in methanol for 10 minutes and rinsed in distilled water. Further, antibody staining and protein blocking was performed by incubating with primary anti-human CD-31 antibody diluted to 1:250 in Dako antibody diluent (Dako North America, Inc.) with background reducing agents for 30 minutes followed by incubatione with biotinylated goat anti-rabbit diluted 1:200 in protein block (Dako North America, Inc.) for 30 minutes Later, sections were incubated in Vector RTU ABC Elite complex (Vector Laboratories, Inc., U.S. Headquarters, Burlingame, CA, USA) for 30 minutes, rinsed in wash buffer, followed by water. At this point, the sections were incubated in 3,3’-Diaminobenzidine (DAB) (Dako North America, Inc.) for 5 minutes and counterstained in hematoxylin for 30 seconds. Finally, the stained sections were rinsed in running tap water and treated with 1% Ammonium Hydroxide solution. The sections were then dehydrated, cleared and mounted in the same manner as discussed in above histological staining procedures.