Tissue Microarray and Immunofluorescence Staining

Human paraffin embedded tissue microarray (TMA: #SK2444A and #SKN1001, see Supplementary Table S4 for details) were purchased from US Biomax (Derwood, MD, USA). Antigen retrieval (10 mM sodium citrate buffer, 0.05% Tween20) was performed at 95 °C for 20 min before immunostaining. RHE were prepared for immunofluorescence as previously described [6 (link)]. The following steps are similar for both TMA and RHE sections. After dewaxing and rehydration, tissue sections were permeabilized using PBS 0.1% Triton X-100, 0.1 M glycine during 10 min at room temperature (RT). Samples were then blocked with PBS containing 5% goat serum, 2% BSA and 0.1% Tween20 for at least 1 h at RT. After washing steps, primary antibodies were incubated overnight at 4 °C (Supplementary Table S2). After washings, secondary antibodies were incubated 45 min at RT and nuclear staining was performed using ProLong^TM Glass Antifade Mountant with NucBlue^TM (Thermo Fischer Scientific, #P36981). Negative controls were performed by omitting primary antibodies. Images were visualized using High Content Screening Yokogawa CQ1 microscope (Yokogawa, Tokyo, Japan), digitalized using sCMOS camera (Olympus, Hamburg, Germany) and analysed using ImageJ software (version 2.1.0/1.53c).