Tissue samples from Wistar rat myocardium were obtained and processed for ultrastructural investigation as previously described [28 (link)].
Ten Wistar rats, having a body weight of 200–250 g, with free access to food and water, maintained in a temperature-controlled facility with a 12-hrs light/dark cycle were used for this study. All animal experiments have been carried out in accordance with the ethical Guidelines for Animal Experimentation and the study was approved by the Bioethics Committee of ‘Carol Davila’ University of Medicine Bucharest.
Ventricular and atrial myocardium was harvested under anaesthesia after perfusion-fixation (1.5% buffered glutaraldehyde) followed by immersion in 4% buffered glutaraldehyde. Tissue samples were cut into 1 mm three small fragments and fixed for 4 hrs in 4% glutaraldehyde in 0.1M cacodylate buffer, pH 7.4 at 4'B0C. The fragments were post-fixed for 1 hr in buffered 1% OsO4, dehydrated in an ethanol series and then processed for Epon 812 embedding at 60'B0C for 48 hrs.
One-micron-thick sections stained with 1% toluidine blue were examined for a precise orientation of the subsequent thin sections. The ultrathin sections were cut using an LKB ultramicrotome with a diamond knife and double stained with 1% uranyl acetate and Reynolds lead citrate.
Electron microscopy examination was performed with both a Philips CM 12 and a Philips 301 transmission electron microscope at 60 kV. The images were recorded with Morada 11 megapixel CCD camera and analysed with iTEM SYS software. Data are expressed as mean ‘B1 SD. Digitally colour images were obtained using Adobe Photoshop software.
Ten Wistar rats, having a body weight of 200–250 g, with free access to food and water, maintained in a temperature-controlled facility with a 12-hrs light/dark cycle were used for this study. All animal experiments have been carried out in accordance with the ethical Guidelines for Animal Experimentation and the study was approved by the Bioethics Committee of ‘Carol Davila’ University of Medicine Bucharest.
Ventricular and atrial myocardium was harvested under anaesthesia after perfusion-fixation (1.5% buffered glutaraldehyde) followed by immersion in 4% buffered glutaraldehyde. Tissue samples were cut into 1 mm three small fragments and fixed for 4 hrs in 4% glutaraldehyde in 0.1M cacodylate buffer, pH 7.4 at 4'B0C. The fragments were post-fixed for 1 hr in buffered 1% OsO4, dehydrated in an ethanol series and then processed for Epon 812 embedding at 60'B0C for 48 hrs.
One-micron-thick sections stained with 1% toluidine blue were examined for a precise orientation of the subsequent thin sections. The ultrathin sections were cut using an LKB ultramicrotome with a diamond knife and double stained with 1% uranyl acetate and Reynolds lead citrate.
Electron microscopy examination was performed with both a Philips CM 12 and a Philips 301 transmission electron microscope at 60 kV. The images were recorded with Morada 11 megapixel CCD camera and analysed with iTEM SYS software. Data are expressed as mean ‘B1 SD. Digitally colour images were obtained using Adobe Photoshop software.
