Mouse igg1 isotype control

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).

Flow cytometry for CEACAM1, 3, 5, 6, 8 and 21 was performed as described^{10 (link),17 (link)}. Briefly, melanoma cells were stained with mouse anti-human PE-labeled CEACAM1/CD66a (R&D, Wiesbaden, Germany), PE-labeled anti-CEACAM3 (Sino Biological, Beijing P.R. China), FITC-labeled anti-CEACAM5 (Bio-Rad/AbD Serotec, Munich, Germany), APC-labeled anti-CEACAM6 (R&D, Wiesbaden, Germany), FITC-labeled anti-CEACAM8 (Miltenyi, Bergisch-Gladbach, Germany), FITC-labeled anti-CEACAM21 (antibodies online.com, Aachen, Germany) or the corresponding isotype controls (Miltenyi). For CEACAM3,5,6,8,21 cells were also fixed (4%PFA) and permeabilized (1% Saponin) prior to staining. IGFBP7 was demonstrated by incubation with anti-human IGFBP7 (ab89347, Abcam, Cambridge, United Kingdom) or corresponding mouse IgG1 isotype control (Dako, Hamburg, Germany) followed by staining with allophycocyanin-labeled goat anti-mouse Ig (BD, Heidelberg, Germany). Intracellular Latexin was shown by serial incubations of paraformaldehyde fixated cells with goat anti-Latexin (AF3246, R&D) or corresponding goat isotype control (Dako) followed by biotinylated rabbit anti-goat (Dako) and allophycocyanin-labeled streptavidin (BD). Stained cells were subjected to fluorescence assisted flow cytometry on a FACSCalibur (BD). Files were analyzed using Win MDI 2.9 software.