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.