Ventana benchmark special stain platform

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.

Immunohistochemical staining (IHC) of Ki-67 protein or FGF11 was performed on 4-mm sections of formalin-fixed paraffin-embedded (FFPE) tumor tissue using VENTANA BenchMark Special Stain platform (Roche, Indianapolis, IN, USA) based on the manufacturer’s instructions. Antibody used for Ki-67 IHC staining was Anti-Ki67 antibody (ab15580, 0.5 µg/ml in TBST, Abcam, USA). Antibody used for FGF11 IHC staining was Anti-FGF11 antibody [MM0282-6J20] (ab89713, 0.5 µg/ml in TBST, Abcam, USA).
Hematoxylin and Eosin (H&E) staining was performed using H&E Stain Kit (ab245880, Abcam, USA). Deparaffinized/hydrated section was incubated in adequate Hematoxylin solution for 5 min. The section was rinsed twice with distilled water to remove excess stain. Then adequate Bluing Reagent was applied to completely cover tissue section and incubate for 30 secs. After washing with distilled water, the section was dehydrated in absolute alcohol, followed by staining with Eosin Y Solution to completely cover tissue for 2–3 min. The section was rinsed using absolute alcohol for three times and mounted in synthetic resin for observation.