Heart tissue was embedded in tissue freezing media (Triangle Biomedical Science) snap-frozen in liquid nitrogen and sectioned in the coronal plane using Leica CM1900 cryostat (Leica Microsystems, Bannockburn, IL, USA). Coronal and axial tissue sections (5 to 7 μm thickness) were mounted on positively charged glass slides and fixed/permeabilized in a 1:1 mixture of acetone and ethanol. Sections were blocked with 3% BSA in PBS and incubated with primary antibodies against α-MHC (Abcam), HDAC1, HDAC2, α-SMA (Abcam), CD90 (BD), and Vimentin (Abcam). Specific staining was visualized using corresponding secondary antibodies conjugated with Alexa 488 or 568 (Molecular Probes). Nuclei were stained with DAPI, 4′ 6-diamidino-2-phenylindole (Life Technologies).
Cells were fixed/permeabilized in a 1:1 mixture of acetone and ethanol. Cells were blocked with 3% BSA in PBS and stained with primary antibodies. Corresponding secondary antibodies were conjugated with Alexa-488 or Alexa 568 (Molecular Probes). Nuclei were stained with DAPI, 4′ 6-diamidino-2-phenylindole (Life Technologies).
Fluorescent images were captured using Leica TCS SPE confocal system configured with Leica DM 2500 microscope. Excitation maximums of 488 nm, 532 nm, and 405 nm, were used for image acquisition. Images were processed using LAS AF software (Leica Microsystems).
Cells were fixed/permeabilized in a 1:1 mixture of acetone and ethanol. Cells were blocked with 3% BSA in PBS and stained with primary antibodies. Corresponding secondary antibodies were conjugated with Alexa-488 or Alexa 568 (Molecular Probes). Nuclei were stained with DAPI, 4′ 6-diamidino-2-phenylindole (Life Technologies).
Fluorescent images were captured using Leica TCS SPE confocal system configured with Leica DM 2500 microscope. Excitation maximums of 488 nm, 532 nm, and 405 nm, were used for image acquisition. Images were processed using LAS AF software (Leica Microsystems).