Histomark red

Paraffin-embedded tumor sections were cut (4 µm), deparaffinized, rehydrated and heated in sodium citrate solution at pH 6.0 for epitope retrieval. 0.5% Triton X-100 was used for permeabilization and 1% bovine serum albumin and 5% goat serum for blocking. Primary antibody staining was carried out with anti-caspase-3 (cleaved) (Cell Signalling, #9661) at 4 °C overnight. This was followed by treatment with biotin-conjugated polyclonal swine anti-rabbit secondary antibody (E0353, Agilent Technologies, Santa Clara, CA, USA) and phosphatase-conjugated streptavidin (Seracare Life Sciences, Milford, MA, USA). For visualization, HistoMark^® RED (Seracare Life Sciences) and hematoxylin counterstaining (Sigma-Aldrich) were performed. Quantification was done with ImageJ (Fiji) freeware using the color threshold tool. Caspase-positive cells were counted per area.

Tissue sections were prepared and treated as described for immunofluorescence staining. Staining was performed with anti-Ki67 primary antibody (9027, Cell Signaling) at 4 °C overnight followed by biotin-conjugated polyclonal swine anti-rabbit secondary antibody (E0353, Agilent Technologies, Santa Clara, CA, USA) and phosphatase-conjugated streptavidin (Seracare Life Sciences, Milford, CT, USA). Signals were visualized by HistoMark^® RED (Seracare Life Sciences) and hematoxylin counterstaining (Sigma-Aldrich). For quantification, Ki67-positive cells were counted per high power field (HPF = 0.159 mm², 10 HPF counted). The mean of Ki67-positive cells was calculated and expressed in percent.