C2821

Six days old cultured normal myotubes were washed with 1x PBS, fixed in 4% paraformaldehyde both supplemented with 100 µM N-benzyl-p-toluene sulphonamide (BTS) and blocked by incubating in 5% goat or rabbit serum, as described in detail^{34 (link)}. Primary and secondary antibodies, diluted in PBS supplemented with 0.2% BSA and Triton X-100 were as follows: mouse monoclonal antibody 1A against DHPRα_1S (1:2,000, MA3-920, Affinity Bioreagents), rabbit polyclonal anti-ANO1 (1:50, ab84115, Abcam), goat polyclonal anti-ANO5 (1:500, sc-169628, Santa Cruz), rabbit anti-GFP (1:5,000, A11122, Invitrogen), secondary goat anti-mouse Alexa Fluor 594, goat anti-rabbit Alexa Fluor 488 (both 1:4,000, A11032 and A11034, respectively, Invitrogen), rabbit anti-goat Cy3 and rabbit anti-mouse FITC (1:500, C2821, and 1:2,000, F9137, respectively, Sigma).
Images were recorded with a cooled CCD camera (Diagnostic Instruments) and MetaVue image processing software (v 6.2, Universal Imaging, PA). Quantification of ANO1 surface membrane expression after siRNA knock-down was determined by measuring the average fluorescence intensity along the periphery of CFP positive myotubes, obtained from at least two different cultures using the MetaVue software.

Explanted septal, left-, and right–ventricular myocardial tissue was fixed in 4% Roti Histofix (Carl Roth, Karlsruhe, Germany) and was embedded in paraffin. We prepared 5 µm sections using a microtome (Leica, Wetzlar, Germany) that were deparaffinized using xylene and ethanol as described [25 (link)]. Bovine serum albumin (5% in phosphate buffered saline, PBS) was used for blocking (30 min, room temperature). Polyclonal goat anti-desmin antibodies (15 µg/mL, #AF3844, R&D Systems, Minneapolis, MN, USA) were used in combination with secondary anti-goat antibodies conjugated to Cy3 (1:100, #C2821, Sigma-Aldrich, St. Louis, MO, USA) for desmin labelling. We used 4′,6-diamidino-2-phenylindole (DAPI, 1 µg/mL) for nuclei staining (5 min, RT). Myocardial tissue was embedded using Fluorescent Mounting Medium (Dako, Glostrup, Denmark). Confocal microscopy was performed as previously described [26 (link)].