Chicken anti cytokeratin 8 18

Cells were seeded onto glass cover slides and allowed to attach for 24 h. Medium was removed and cells fixed with 4% paraformaldehyde for 10 min. After fixation, cells were washed with PBS and permeabilized with 0.2% Triton^®-X-100 (Serva, Heidelberg, Germany) for 5 min. Subsequently, cells were incubated in StartingBlock^TM (PBS) blocking buffer (ThermoFisher Scientific, Waltham, MA, USA) for 1 h at room temperature. The following primary antibodies were added for 1 h at room temperature: rabbit anti HIF-1α (dilution 1:50; EP1215Y, Abcam, Cambridge, UK) and chicken anti cytokeratin 8/18 (dilution 1:100; Sigma-Aldrich). After removal of primary antibodies, cover slides were washed with PBS and secondary antibodies added: goat anti rabbit 555 (1:200, Invitrogen) and donkey anti mouse 488 (1:200, Invitrogen). Subsequently, slides were mounted in Vectashield mounting medium containing DAPI (Vector Laboratories, Burlington, CA, USA). Cells were visualized using fluorescent microscopy on a Zeiss Axio Imager Z2. HIF-1α staining intensity was scored using TissueQuest software (TissueGnostics, Vienna, Austria) and normalized to DAPI positive cells.

Cells were seeded onto glass coverslips and allowed to attach for 48 hours. Frozen tissue sections (6 μm) or cells on slides or coverslips were washed with PBS and fixed with 4% paraformaldehyde for 10 min. Subsequently samples were washed with PBS and permeabilized with PBS supplemented with 1% bovine serum albumin (BSA) and 0.2% Triton X-100 for 5 min. After a 30-min blocking step with PBS containing 1% BSA, samples were incubated with the antibody MTC02, which recognizes a mitochondrial protein of 60 kDa (dilution 1:200 for cells, 1:100 for tissue; Abcam, Cambridge, UK) and chicken anti cytokeratin 8/18 (dilution 1:300; Sigma) for 1 hour at 37°C. One sample of cells or tissue was incubated with mouse IgG1 isotype control (Dako) instead of primary antibody at the same concentration. After washing, coverslips were incubated with goat anti-mouse and goat anti-chicken fluorescently-labeled secondary antibodies (1:500; Life technologies, Carlsbad, CA). Samples were finally washed and mounted with Vectashield Hard Set mounting medium containing DAPI (Vector Laboratories, Burlingame, CA). Cells and mitochondria were visualized using fluorescent microscopy on a Zeiss Axio Imager M1. For quantification of MTCO2 staining, slides were scored automatically by using TissueQuest software (TissueGnostics, Vienna, Austria).