Anti ki 67 clone d3b5

Cells were fixed with 4% paraformaldehyde for 10 min, permeabilised with 0.1% Triton X-100 (Sigma, St Louis, MO, USA) diluted in PBS solution for 10 min, followed by blocking in 2% bovine serum albumin (Roche Diagnostic) for 2 h at room temperature. Cells were then stained overnight at 4°C with the following primary antibodies diluted in blocking solution: anti-AQP5 1:100 (clone EPR3747; Abcam, Cambridge, UK), anti-pro-SPC 1:1000 (Millipore, Burlington, MA, USA), anti-p21 1:100 (clone C-19; Santa Cruz Biotechnology, Dallas, TX, USA) and anti-Ki-67 (clone D3B5; Cell Signaling Technology, Danvers, MA, USA). Cell were washed with PBS and incubated for 2 h with the respective secondary antibodies conjugated to Alexa Fluor 594 (Thermo Fisher Scientific). Cell nuclei were counterstained with Hoechst 33342 (Thermo Fisher Scientific).

Multiplex immunohistochemistry was carried out as described previously
¹⁸ (link) using an Opal Automation IHC Kit (Akoya Bioscience) and Bond RXm autostainer (Leica Biosystems). The sections were stained with DAPI and the following Abs: anti‐CD45 (clone D3F8Q; Cell Signaling Technology; 1:500), anti‐DIG (clone 9H27L19; Thermo Fisher Scientific; 1:500), anti‐Ki‐67 (clone D3B5; Cell Signaling Technology; 1:500), and anti‐pCK (rabbit poly; Bioss Antibodies; 1:250). Stained slides were mounted with ProLong Diamond (Thermo Fisher Scientific) and imaged with a Mantra Quantitative Pathology Workstation (Akoya Biosystems). The obtained images were analyzed with inForm Tissue Finder software (Akoya Biosystems). To calculate the percentage of Ki‐67 positive cancer cells, inForm software was trained to detect tissue and cell phenotypes using machine‐learning algorithms based on the following criteria: areas with pCK expression = tumor, other areas = stroma, pCK+ CD45− cells = cancer cells, pCK− CD45+ = blood cells, and pCK− CD45− = other cells. inForm software computed the percentage of Ki‐67‐positive cells among cancer cells. The average percentage was calculated from five images for each specimen.