Metavue image processing software

Immunofluorescence analysis was performed as described (15 (link)) using the following antibodies: rabbit polyclonal anti-GFP (1:4,000; Molecular Probes, Eugene, OR); mouse monoclonal anti-RyR (34-C; 1:1000; Alexis Biochemicals, Lausen, Switzerland); secondary goat-antimouse Alexa-594 and goat-antirabbit Alexa-488 (1:4,000; Molecular Probes). Images were captured on a Zeiss Axiophot microscope with a cooled charge-coupled device camera and METAVUE image-processing software (Universal Imaging, West Chester, PA).

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.