Photometrics coolsnap digital camera

BrdU immunohistochemistry was performed as described^{26 (link)}. Paraffin sections were dewaxed and underwent antigen retrieval, followed by denaturation with 0.1 N HCl for 45 min. The sections were then quenched with 3% hydrogen peroxide for 10 min, blocked with 5% BSA/PBS for 1 h, and incubated with sheep anti-BrdU antibody (1:500 dilution; Abcam) or isotype control at 4 °C overnight. On the next day, the sections were incubated with donkey anti-sheep biotinylated secondary antibodies (1:400 dilution; Abcam) for 1 h and then with the Vectastain ABC reagent (Vector Laboratories) for 30 min. BrdU positive staining was revealed by DAB solution (Dako) under a Zeiss Axioskop II microscope (Carl Zeiss). The sections were counterstained with the Mayer’s hematoxylin, dehydrated, and mounted using aqueous mounting medium (Dako). Images were acquired using a Photometrics CoolSNAP digital camera (Roper Scientific).

Immunohistochemical analysis of Hypoxyprobe (pimonidazole) were conducted according to the manufacturer’s instructions. The paraffin sections (5 µm) were deparaffinized and dehydrated. Antigen retrieval was performed using antigen retrieval buffer (Dako, Hamburg, Germany) in a microwave oven, followed by incubation with 3% H₂O₂ for 10 min at room temperature. To reduce nonspecific staining, the sections were blocked with 5% BSA for 30 min at 37 °C, then incubated with pimonidazole primary antibody (1:50 dilution, mouse Mab, Burlington, MA, USA) at 4 °C overnight. The following day, the sections were incubated with polyclonal goat anti-mouse biotinylated secondary antibodies (1:200 dilution, Dako, Hamburg, Germany) for 60 min, and Vectastain ABC Standard Kit reagents (Vector Laboratories, Burlington, MA, USA) for another 30 min. A diaminobenzidine (DAB) kit (Dako, Hamburg, Germany) was applied for color development and monitored under a Zeiss Axioskop II microscope (Carl Zeiss, Munich, Germany). The sections were counterstained with the Mayer’s hematoxylin for 1 min, dehydrated and mounted using aqueous mounting medium (Dako). Images were captured using a Photometrics CoolSNAP digital camera (Roper Scientific, Trenton, NJ, USA). All incubations were performed at room temperature and washes with PBS were conducted between each step.