Anti ki 67 d3b5

For Ki-67 staining, formalin-fixed paraffin-embedded tissue sections were de-waxed and rehydrated, incubated in Diva Decloaker antigen retrieval solution (Biocare) and boiled for 20 min in a pressure cooker. Peroxidase activity was blocked using the Enzyme Block for 15 min (DAKO). Slides were stained with 1:400 anti-Ki-67 (D3B5; Cell signaling) for 1 h followed by another hour of incubation with 1:500 of goat anti-rabbit (Jackson laboratories). Finally, slides were incubated with DAKO DAB substrate (DAKO) for 10 min and counter-stained with hematoxilin, then mounted. For Alcian Blue staining (1% solution, Sigma), slides were stained at room temperature for 30 min and then washed. Cell nuclei were stained with Kernchtrot Nuclear Fast Red stain (1 min). Slides were washed with distilled water, dehydrated with anhydrous alcohol and mounted.

Immunohistostaining of Ki67 was performed on 4-mm sections of formalin-fixed paraffin-embedded (FFPE) tumor tissue using VENTANA BenchMark Special Stain platform (Roche, Indianapolis, IN, USA). Briefly, the section was dehydrated and antigen unmasking was performed by heating the section submersed in 1X citrate unmasking solution (SignalStain® Citrate Unmasking Solution, Cell Signaling Technologies, #14,746) for 10 min at a sub-boiling temperature (95°-98°C). After cooling, sections were washed in dH₂O three times and then incubated in 3% hydrogen peroxide for 10 min. The section was blocked 1 h at room temperature in TBST buffer with 5% Normal Goat Serum, and then incubated with primary antibody anti-Ki-67 (D3B5) (Cell Signaling Technologies, #9129) diluted 1:500 in SignalStain® Antibody Diluent (#8112) overnight at 4°C. After 3 washes with TBST buffer, the section was incubated with HRP-conjugated secondary antibody (1:3000 dilution; Cell Signaling Technologies, #7074) for 60 min at room temperature. After wash, the section was stained by a diaminobenzidine staining kit (ZSGB-BIO, Beijing, China) for 30 min at room temperature. The section was washed in dH₂O two times and then dehydrated. Section was mounted with coverslips using the mounting medium (Cell Signaling Technologies, #14,177) before imaging.