Diaminobenzidine dab kit

Liver tissues were fixed for 24 h in 10% formalin, embedded in paraffin, and cut into 4 μm sections. Each section was incubated overnight at 4°C with an anti-PCNA antibody (1 : 500 dilution, Santa Cruz Biotechnology, USA) followed by incubation with a horseradish peroxidase- (HRP-) conjugated goat anti-mouse antibody (Dako, Denmark). A diaminobenzidine (DAB) kit (Dako, Denmark) was used for immunohistochemical visualization. Sections were visualized at 200x magnification, and PCNA staining intensity was analyzed in six random fields per section using Image-pro Plus 6.0 software. Each sample was analyzed in triplicate.

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.