600 electron microscope

Liver pathology was evaluated using conventional histological analysis on hematoxylin and eosin (H & E) stained sections. Further evaluation of pathology was achieved by high-resolution light microscopy using glutaraldehyde-fixed, osmium tetroxide post-fixed, epoxy-embedded, and toluidine blue-stained sections (11 (link), 13 (link)) and electron microscopy. Liver fibrosis was evaluated by Sirius red staining of formalin-fixed, paraffin-embedded liver sections for visualization of collagen. Accumulation of intrahepatic fat and collagen content was quantified by computerized densitometry using the ImagePro Plus software (11 (link)–13 (link)). Further evaluation of liver fibrosis was assessed by measuring transforming growth factor alpha (TGF- α) and beta (TGF- β) (18 (link), 19 (link)) by quantitative RT-PCR.For electron microscopic studies, thin sections from selected tissue blocks were sectioned with an LKB ultramichrotome, stained with uranyl acetate, and examined with a Hitachi 600 electron microscope (Hitachi, Indianapolis, USA), as described before (11 (link), 13 (link)).

All mice were euthanized with a lethal intraperitoneal injection of sodium pentobarbital (200 mg/kg body weight). Blood samples were collected from each animal by cardiac puncture immediately after death, and serum was separated and stored at −20°C for subsequent testosterone assay. The gastrocnemius muscles from each mouse were removed and weighed. Portions of the tissues were snap frozen in liquid N₂ and stored frozen for subsequent analysis by Western blotting. Additional portions from each mouse were either fixed in 2.5% glutaraldehyde for electron microscopy or 4% formalin for histological and immunohistochemical studies. Portions of glutaraldehyde fixed muscles were further diced into small pieces, post-fixed into 1% osmium tetroxide and embedded in Epon 812 as described previously (Sinha-Hikim et al. 2007 ). Thin sections from selected tissue blocks were cut with an LKB ultramichrotome, stained with uranyl acetate and lead citrate, and examined with a Hitachi 600 electron microscope (Hitachi, Indianapolis, IN). The rationale for using gastrocnemius muscles was based on the results of several earlier studies, which show that this muscle exhibits a substantial decline in mass with age (Martin et al. 2007 ; Braga et al. 2008 (link); Kovacheva et al. 2010 (link); Sinha-Hikim et al. 2013 (link)).