Isolation, culture, and immunostaining of neonatal rat ventricular myocytes

Figures

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  Figure 1. Enzymatic digestion of cardiac tissue and Percoll gradient separation (A and B) The heart ventricles were minced in 4–6 pieces and transfer to a bottle containing 10 mL of Collagenase Buffer. (C) For optimal digestion, the enzymatic solution with the cardiac tissue was placed under agitation at 37°C, 180 rpm. (D) After ∼60 min, the cardiac tissue is almost complete digested. (E–G) Cardiomyocyte enrichment step by discontinuous Percoll gradient centrifugation. (H) After 16 h in culture, check the cells under microscopy for shape, adhesion and spontaneous contractions (see Methods video S2) (40× magnification).

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  Figure 2. Immunofluorescence staining and imaging (A) Fix the cells with 4% paraformaldehyde in PBS. (B) Blocking any unspecific antibodies binding by incubating the cell with 1% BSA, 50 mM glycine in PBS. (C) Incubate with primary antibody. (D) Incubate the cells with a secondary antibody. (E) Alternatively, incubate the cells with phalloidin-rodhamin and DAPI. (F) Mounting the coverslip using mounting medium Vectashield. (G) Representative images of cardiomyocytes staining with phalloidin in green and Dapi in blue. (H) Representative pictures of cardiomyocytes staining with α-actinin antibody in red and Dapi in blue. (I) Representative images of cardiomyocytes stained with MEF2 antibody in green, phalloidin in red and Dapi in blue. Scale bar, 10 μm.