Cardiomyocyte Immunofluorescence and Morphometry

Immunofluorescence staining and cell surface area measurements were performed as detailed earlier [34 (link)]. Briefly, cardiomyocytes cultured on coverslips in 12x well culture plates, either transduced with Cfl2 overexpression/knockdown adenoviral particles, or transfected with miR-301a mimic/inhibitor, were washed 2x with PBS and fixed with 4% paraformaldehyde for 10 min. Fixed cells were washed 2x with PBS followed by a common step of permeabilization and blocking with 0.1% Triton X-100 in 2.5% BSA for 1 h at room temperature. Cells were then incubated for 1 h with primary anti–α-actinin antibody (1:200; Sigma-Aldrich), 5x washes with PBS, followed by the incubation with respective secondary antibody conjugated to Cy3 (Dianova) and DAPI for nuclear staining. After washings with PBS, coverslips were mounted on glass-slides using Fluoromount (Biozol). Immunofluorescence images were captured using BZ-9000 microscope (Keyence). Cell surface area was measured using HybridCellCount module BZ-II Analyzer software (Keyence).