Ultramicrotome

Transmission Electron Microscopy (TEM) was performed at the TEM Queen Mary University London Facility. In short, A7r5 cells were fixed for 2 h in 4% glutaraldehyde solution in 0.1 M pH 7.4 Sørensen phosphate buffer. Next the cells were scraped, resuspended in agarose, and cut in 1mm² cubes when set and stained. Ultrathin sections were cut using Reichert‐Jung Leica Ultramicrotome with a Diatome diamond knife. Sections were imaged using JEOL JEM1400 Transmission Electron Microscope, equipped with TEM Morada camera and iTEM (EMSIS) software.

Cultured cells were harvested and seeded onto 10-cm culture dishes for 24 h, and fixed with 2.5% glutaraldehyde (EM grade) in 0.1 M phosphate buffer pH 7.4 at room temperature for 1 h. The fixed cells were collected by using a cell scraper, washed several times in 0.1 M phosphate buffer, and post fixed in 1% osmium tetroxide. Samples were dehydrated in ethanol (30%, 50%, 70%, 80%, 95%, and 100%), embedding in EMbed 812 Resin (Electron Microscopy Sciences, 14120) and polymerized. In all, 70–90-nm sections were cut on a Reichert-Jung ultramicrotome using a diamond knife (DiATOME). Sections were collected on copper grids, stained with UranyLess and lead citrate, and imaged with a Hitachi H-7000 TEM.

Murine hearts were cut into pieces and fixed in 2% glutaraldehyde for at least 24 h. After immersed in 2% osmium tetroxide and 1% aqueous uranyl acetate for 1 h, and washed with a series of ethanol solutions (50, 70, 90, and 100%), tissues were transferred to propylene oxide, incubated in a mixture of propylene oxide and EMbed 812 for 1 h and then polymerized at 70°C. Sections were cut into 75–80 nm using a Leica ultramicrotome equipped with a Diatome diamond knife and collected onto 200-mesh copper grids. Following poststained in 5% uranyl acetate for 10 min and in Reynold’s lead citrate for 5 min, sections were observed under a 40–120 kV transmission electron microscope (Hitachi H600 Electron Microscope, Hitachi, Japan).

Murine heart tissues were fixed in 2% glutaraldehyde for at least 24 h. Tissues were then immersed in 2% osmium tetroxide and 1% aqueous uranyl acetate, each for 1 h. After being washed with a series of ethanol solutions (50, 70, 90, and 100%), tissues were transferred to propylene oxide, incubated in a 1:1 mixture of propylene oxide and EMbed 812 (Electron Microscopy Sciences) for 1 h and then placed in a 70°C oven to polymerize. Sections (75–80 nm) were cut using a Leica ultramicrotome equipped with a Diatome diamond knife and collected on 200-mesh copper grids. After being post-stained in 5% uranyl acetate for 10 min and in Reynold's lead citrate for 5 min, sections were observed using a 40-120 kV transmission electron microscope (FEI TECNAI G2 Spirit Biotwin, Hong Kong, China). For the in vitro study, primary cardiomyocytes were collected by centrifuging at 1,000 rpm for 10 min and fixing them in 100 μL of 2% glutaraldehyde. The subsequent operations were performed as described above (Hu et al., 2017 (link)).

The cells were fixed in 2.5% glutaraldehyde, 3% paraformaldehyde with 5% sucrose in 0.1 M sodium cacodylate buffer (pH 7.4), pelleted, and post fixed in 1% OsO₄ in veronal-acetate buffer. The cells were stained en block overnight with 0.5% uranyl acetate in veronal-acetate buffer (pH 6.0), then dehydrated and embedded in Embed-812 resin. Sections were cut on a Leica ultra-microtome with a Diatome diamond knife at a thickness setting of 50 nm, stained with 2% uranyl acetate, and lead citrate. The sections were examined using a Hitachi 7800 and photographed with an AMT ccd camera. To quantify the euchromatin content of nuclei, we used ImageJ software. First, images were converted to 8-bit format, then nuclear area was manually selected, and the threshold was adjusted (using IsoData auto setting).