Exciter laser scanning microscope

Images were obtained at room temperature on a Zeiss microscope (Axiovert 200M) equipped with LD A‐plan 20×/0.85 ph1, 10×/0.30ph1 and 40×/.075ph1 objectives or on a Zeiss Exciter laser scanning microscope (LSM) with 40×/1.1 W Corr LD C‐Apochromat objective (Carl Zeiss Ltd (https://www.zeiss.co.uk/), Cambridge, United Kingdom). Images were quantified with image J software (NIH). Neonatal MyHC labeling was quantified with R software. Values are mean ± SEM with a Student's t test (paired or unpaired as appropriate) or ANOVA with a post‐hoc Tukey's test for >2 groups.

An upright Zeiss Exciter laser scanning microscope (LSM) with a 40×/1.1 W Corr LD C-Apochromat objective, and an inverted Zeiss 710 LSM with a 20×/1.0 W Plan-Apochromat and a 40×/1.3 Plan-Apochromat objective were used for FRAP and Z-stacks. Acquisition and maximum intensity projections were made with ZEN 2009/2010 (Zeiss, Jena, Germany). Volocity version 6.0.1 (PerkinElmer, Waltham, MA, United States) was used for XYZ projection, to which a fine Gaussian filter was applied and brightness corrected for visualization purposes only. Images were uniformly contrasted with Adobe Photoshop CS4. Illustrations were made in Adobe Illustrator CS3. GraphPad Prism 6 was used for statistical analysis and graph plotting.

For fluorescence microscopy, cells were grown on coverslips and fixed for 20 min in 2% paraformaldehyde, quenched with 3% BSA, permeabilized with 0.1% saponin (Sigma-Aldrich), and incubated serially with the indicated primary and corresponding secondary antibodies. Images were taken with an Axiovert 200 M/ApoTome microscope and a confocal Exciter laser scanning microscope (Zeiss)^{30 (link)}. Colocalization was measured using AxioVision colocalization and Zen 2009 software (Zeiss). Pearson coefficients were calculated using the CoLocalizer Express software (CoLocalization Research Software).