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Increased Apoptosis Occurring During the First Wave of Spermatogenesis: MATERIALS AND METHODS(3)

METHODS(3)Electron Microscopy

In preparation for electron-microscopic examination, testes were fixed by immersion in 5% glutaraldehyde in s-collidine buffer (0.16 M, pH 7.4) at 20°C. After 30 min of such treatment, the tissue samples were cut into approximately 1-mm3 cubes and thereafter reimmersed in the same fixative for an additional 2 h. Postfixation was performed with 1% osmium te-troxide in 1.5% aqueous potassium ferrocyanide, and the samples were then embedded in epoxy resin (Glycidether 100; Merck, Darmstadt Germany). Ultrathin sections (thickness, 70 nm) were prepared (Reichert E Ultramicrotome; Reichert Jung, Vienna, Austria), stained with uranyl acetate and lead citrate, and finally examined under a JEOL 100 SX electron microscope (JEOL, Tokyo, Japan). can i get antibiotics online

Western Immunoblot Analysis

Total testicular protein was extracted employing modified RIPA buffer (1% NP-40; 0.25% sodium deoxycholate; 150 mM NaCl; 1 mM EDTA; 1 mM PMSF; 1 |xg/ml each of aprotinin, leupeptin, and pepstatin; 1 mM Na3VO4; and 1 mM NaF in 50 mM Tris-Cl, pH 7.4) and quantitated using the Bradford procedure (Bio-Rad, Hercules, CA). This protein fraction (30 l^g of total protein for Bax, Bad, and PARP; 40 |xg for caspase 3; and 60 l^g for Bcl-2) from each animal was subjected to SDS-PAGE on 12% gels (8% in the case of PARP) under reducing conditions. Subsequently, the protein bands thus resolved were transferred electrophoretically to poly-vinylidene fluoride membranes that had been preblocked overnight at 4°C in Tris-buffered saline (TBS; 150 mM NaCl in 10 mM Tris, pH 7.5) containing 5% nonfat dry milk.