A New Staining Method (oto) for Enhancing Contrast of Lipid-containing Membranes and Droplets in Osmium Tetroxide-fixed Tissue with Osmiophilic Thiocarbohydrazide (tch)

Al though the in t roduct ion of fixation of tissue with osmium tetroxide by Palade (8) ushered in the modern era of electron microscopy of biological materials, a need for greater contrast and greater resolution in visualizing membranous structures has s t imulated the in t roduct ion of methods for staining with heavy metal salts, in order to enhance the del ineat ion of the fine archi tecture of cells. This is especially necessary because of the poor contras t of osmium tetroxide-fixed tissue when the epoxy resins are used as embedding materials (9). These methods have relied upon specific affinities of heavy metal salts for various macromolecular components of the cell such as proteins, polysaccharides, nucleoproteins, or l ipoproteins (3, 9, 17, 18). Such staining techniques do not necessarily enhance the staining of the same components of the cell originally stained by the osmium tetroxide, a l though interesting and impor t an t staining effects have been produced thereby. In the course of developing cytochemical methods for electron microscopy, based upon the principle of designing histochemical reagents tha t yield osmiophilic end products (6, 10, l 1), we have discovered a new way to enhance especially the contrast of the lipid components of the cells stained by osmium tetroxide dur ing the initial fixation. O n t r ea tmen t wi th an excess of th iocarbohydrazide (H2NNHCSNHNH2, T C H ) , one end of the molecule at taches to the osmium in the tissue and, when this is followed by exposure to osmium tetroxide, more osmium is bound to these sites. The new method ( O T O ) utilizes this br idging phenomenon (5) and, a l though contrast is increased thereby in all osmiophilic components of tissue, greatest enhancemen t of contrast is produced in tissue components holding the most osmium, i.e., lipid. The O T O method consists of exposing u l t ra th in sections of Araldi te-embedded, osmium tet roxidefixed tissue (8) on gold, stainless steel, or nickel grids, I without a support ing membrane , to a 1 hot aqueous solution of T C H 2 for 1 hr at 50°C, ~ followed by several washes with hot water (initial tempera ture 50°C) for 10 to 15 min to remove unbound TCH. The sections are then exposed to osmium tetroxide again, resulting in fur ther deposition of osmium. The best results are obtained by exposing the sections to osmium tetroxide vapor 4 for 1 hr in a closed vessel suspended in a water ba th at 60°C as described earlier (11), or very good results may be obtained with perhaps a little less contrast and considerably less cost by t rea t ing the grids with a 2 % solution of osmium tetroxide