Neonatal Rat Cardiomyocyte Isolation and Transfection

NRC isolation, culture, transfection, and staining were performed essentially as described previously (Pernigo et al., 2010 (link)). Briefly, NRCs were transfected with GFP-tagged transiently expressing constructs (pEGFPC2-, Clontech Laboratories) using Escort III (Sigma-Aldrich). After 48 hr culture to promote protein expression, cells were fixed with 4% paraformaldehyde/PBS, permeabilized with 0.1% Triton X-100/PBS, and then stained with the appropriate antibodies. The antibodies used for the current work were as follows: MyB4, a mouse monoclonal antibody to the myomesin domain My12 (Grove et al., 1984 (link)); and Ob5859, a rabbit polyclonal antibody to two consecutive Ig domains in obscurin, Ob58 and Ob59 (Fukuzawa et al., 2005 (link), Young et al., 2001 (link)). All fluorescent-conjugated secondary antibodies were purchased from Jackson ImmunoResearch. All images for ratiometry analysis were collected on a Zeiss LSM510 confocal microscope as described previously (Fukuzawa et al., 2008 (link)). Image analysis was carried out as described in our previous work (Pernigo et al., 2015 (link)). Further details are available in the Supplemental Experimental Procedures.