Immunohistochemical staining was performed as described [26 (link)]. Formalin-fixed, paraffin-embedded tissue was cut at 4-μm thicknesses and deparaffinized with xylene and ethanol. Endogenous peroxidase activity was blocked by methanol containing 0.3% hydrogen peroxidase. Antigen retrieval was performed by boiling in a microwave oven (citrate buffer, pH 6.0). The sections were incubated overnight at 4°C with primary antibodies against anti-RIP3 (ab56164; Abcam, Cambridge, UK; 1:100), anti-MLKL (ab194699; 1:100), anti-CXCR2 (ab65968; 1:200), anti-CXCL5 (ab9802; 1:500) and anti-CXCL1 (ab86436; 1:100). The immune complexes were then visualized using Envision Detection System (Dako, California, USA) and 3,3′-diaminobenzidine (DAB) Kit (Dako). DAB intensity of MLKL was evaluated as previously described using ImageJ software [27 ]. Images were adjusted by subtracting background as RGB values close to 255 in empty area. Tumor area was selected and mean intensity of DAB color spectrum was calculated, ranging 0 (black) to 255 (total white). The final DAB intensity was calculated according to the formula f = 255—i, where f = final DAB intensity, i = mean DAB intensity. Intensity was calculated in 5 fields at the borders and cores from each patient at 200× magnification under a light microscope.
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