α ereg

Cells were detached and resuspended in flow cytometry buffer (2/3 (v/v) PBS and 1/3 (v/v) RPMI without additives). For immunostaining, 10 μg/mL α-EREG (goat, polyclonal, R&D Systems, Minneapolis, Minnesota, US) or α-TACE/ADAM17 (mouse, MM0561-8C13, Novus Biologicals, Wiesbaden, Germany) antibody was added to flow cytometry buffer and incubated for 1 h at 4°C. ChromPure goat or mouse IgG (Jackson ImmunoResearch, Cambridgeshire, UK) were used as controls for corresponding primary antibody. After three washing steps, cells were incubated for 1 h at 4°C with FITC-conjugated α-goat (donkey, polyclonal, Jackson ImmunoResearch, Cambridgeshire, UK) or α-mouse (goat, polyclonal, Jackson ImmunoResearch, Cambridgeshire, UK) antibody diluted 1:50 in flow cytometry buffer. Finally, cells were washed three times and resuspended in PI diluted 1:200 in DMEM without additives. The acquisition was performed by FACS Calibur System (Becton Dickinson, Franklin Lakes, NJ, USA) and analyzed by Cell Quest Pro™ Version 6 (Becton Dickinson, Franklin Lakes, NJ, USA). To determine the size and granularity of cells the forward (FSC) and sideward (SSC) scatter were used.

Paraffin‐embedded tissues were deparaffinized with xylene and rehydrated in a descending alcohol series (100%, 96% and 70%). Slices were boiled in citrate buffer (pH = 6), blocked with 1% BSA in PBS (60 min, room temperature) and incubated overnight at 4°C in 0.5% BSA in PBS containing 10 μg/mL α-EREG (goat, polyclonal, R&D Systems, Minneapolis, Minnesota, US) antibody. After three washing steps with PBS, slices were incubated (1 h, room temperature) in 0.5% BSA in PBS with 1:200 diluted biotinylated α-goat (rabbit, polyclonal, Dako, Agilent Technologies, Waldbronn, Germany) antibody. After washing with PBS three times, signal enhancement was achieved by Vectastain^® (Linaris, Wertheim‐Bettingen, Germany) according to the manufacturer’s instructions. Immunoreaction was visualized with diaminobenzidine (DAB) (Sigma-Aldrich, Hamburg, Germany), followed by nuclear staining using hematoxylin solution (Thermo Fisher Scientific, Oberhausen, Germany). Finally, slices were dehydrated and mounted with Xylene Substitute Mountant (Thermo Fisher Scientific, Oberhausen, Germany). Slides were scanned using a Aperio ScanScope Slide Scanner (Leica Biosystems, Nußloch, Germany) and saved as ScanScope Virtual Slide (.svs) files.