Immunostaining
Cells were grown on glass coverslips and fixed with 2 % paraformaldehyde for 5 min (for rabbit-anti-DSG 3-mAb (Biozol, Germany), rabbit-anti-DSG 1-pAb (Abclonal, USA), mouse-anti-DSG 1-mAb (Progen, Germany) mouse-anti-GAPDH-mAb (Santa Cruz, USA), mouse-anti-HSP60-mAb (Thermo Scientific, USA) rabbit-ant-IP3R -pAb (Abcam, USA), mouse-anti-Orai1-mAb (Santa Cruz, USA), rabbit-anti-PI4K a-mAb (Abbexa, United Kingdom), rabbit-Anti-p-PKC α-mAb (Abcam, USA), rabbit-anti-p-PLC γ1-mAb (Cell signaling, USA), rabbit-anti-Stim1-mAb (Cell signaling, USA) and in ethanol (-20 °C) shaking on ice for 30 min and acetone (-20 °C) for 3 min (For mouse-anti-cytokeratin-pan-mAb (Sigma Aldich, USA) 1:100-200. Paraformaldehyde fixed cells were permeabilized with 1 % Triton X-100 in PBS for 5 min (for acetone fixed cells no permeabilization was necessary), the cells were blocked with 3 % bovine serum albumin (BSA) and 1 % normal goat serum in PBS for 30 min. Primary antibodies were applied overnight at 4 °C. Cy3 coupled goat-anti-rabbit/mouse/human secondary antibodies (Dianova, Germany) and Alexa-488-phalloidin (Life technologies, USA) were incubating for 1 h and DAPI 1:10.000 for 15 min. The cover slips were mounted with 2 % n-propyl-gallate and evaluated with a SP5.II confocal microscope with a 63x NA 1.4 PL APO objective (Leica, Germany). After heating to 60 °C for 30 min the skin slices were treated the same, except for 1 h permeabilization. The cells were washed 3x with PBS in between each step except ethanol to acetone.