Hrp linked goat anti rabbit secondary antibody

Five-micrometer sections of formalin-fixed and paraffin-embedded tumor tissue were mounted on charged glass slides and dried at 58 °C for 60 min. Slides were first deparaffinized with xylene then epitope retrieval carried out using citrate buffer in a 2100 Retriever pressure cooker (PickCell Laboratories, San Jose, CA, USA). The sections were blocked with 10% goat serum for 1 h, then incubated with primary antibody (anti-p27, 1 : 50, Abcam (ab7961)) overnight, followed by an HRP-linked goat anti-rabbit secondary antibody (1 : 300, Pierce Biotechnology). The sections were developed with 3,3′-diaminobenzidine tetrahydrochloride (Vector Laboratories, Burlingame, CA, USA) for 2 min and nuclei counterstained with hematoxylin (Sigma-Aldrich, St. Louis, MO, USA) for 2 min. PBS washes were performed between all steps. The slides were dehydrated in graded alcohols (100%, 95% and 80% ethanol (vol/vol) in H₂O), cleared in xylene and coverslips attached with Permount (Fisher Scientific, Waltham, MA, USA). Slides were scanned using a ScanScope XT Digital Slide Scanner (Aperio, Vista, CA, USA) and data were analyzed with the positive pixel count algorithm (ImageScope, Aperio).