Samdri pvt 3d system

Spermatozoa collected from the cauda epididymis (from mice at age of 10–14 weeks) were incubated in Toyoda, Yokoyama, and Hoshi (TYH) medium to induce their dispersion, transferred to a 2.0-ml tube, and washed with 0.1 M phosphate buffer (pH 7.4). They were mounted on coverslips, fixed with 1% glutaraldehyde in phosphate buffer on ice, washed with phosphate buffer, fixed again with 1% OsO₄ in phosphate buffer containing 1% potassium ferrocyanide, and subjected to conductive staining with 1% tannic acid and 1% OsO₄. The specimens were dehydrated with a graded series of ethanol solutions, subjected to critical point drying with the use of a Samdri-PVT-3D system (Tousimis), coated with OsO₄ with an HPC-30W device (Vacuum Device), and observed with an S-4800 field-emission scanning electron microscope (Hitachi).

For SEM, three adult specimens of B. townsendi were dissected immediately after collection while still on board the research vessel, and extracted ICs were fixed in 2.5% glutaraldehyde in Sorenson’s phosphate buffer at pH 7.2. Samples were held in fixative at 4°C for several days, then rinsed three times in the same buffer and postfixed in 1% OsO₄ for 1 h. After the initial osmium treatment, they were rinsed once in buffer, once in buffer diluted to 50% with water, and once more in pure water. Tissues were then subjected to thiocarbohydrazide-mediated osmium binding (Kelley et al., 1975 ): they were immersed in a saturated solution of thiocarbohydrazide for 1 h, rinsed twice with water, then treated with 1% OsO₄ in water for 1 h. Tissues were then rinsed twice in water, dehydrated through an ascending ethanol series (to 100%), critical-point-dried using a Samdri-PVT-3D system (Tousimis Research Corp., Rockville, MD, United States) with CO₂ as the transitional fluid, and mounted on stubs using copper-conductive tape with conductive adhesive on both sides. Specimens were coated with gold/palladium using a Pelco SC-4 sputter-coater (Ted Pella Inc., Redding, CA, United States) and imaged with a FEI Quanta 200 scanning electron microscope (Thermo Fisher Scientific Inc., Waltham, MA, United States).

Tissue samples were prepared with the freeze-fracture method as described previously [3 (link)] with slight modifications. The testes were dissected from the
mice and sliced into rings 5 mm thick using safety razors. The specimens were fixed in 1% OsO₄ in 0.1 M phosphate buffer (pH 7.4) for 1 h at 4°C, immersed in 50% dimethyl
sulfoxide (DMSO) solution, and cracked with a freeze-cracking apparatus TF-2 (Eiko, Tokyo, Japan). After the specimens were rinsed, they were transferred to buffered 0.1% OsO₄ and
left standing for 48–72 h at 20°C. The samples were then postfixed with buffered 1% OsO₄ for 1 h at 4°C, and stained with 2% tannic acid solution (Wako) for 2 h followed by
buffered 1% OsO₄ for 1 h. The specimens were dehydrated in a graded series of ethanol solutions (50, 70, 90, 100, 100, 100%), and critical point dried using a Samdri-PVT-3D system
(Tousimis, Rockland, MD, USA). The specimens were then mounted on sample stages, and coated with osmium using an osmium coater HPC-30W (Vacuum Device, Ibaraki, Japan). The samples were
observed with an S-4800 field emission scanning electron microscope (Hitachi, Tokyo, Japan).

Cauda epididymal spermatozoa were incubated in TYH medium to disperse. Spermatozoa were collected into 2.0 ml tube and washed with 0.1 M phosphate buffer (pH 7.4). Spermatozoa were mounted on cover slips and fixed with 1% glutaraldehyde in 0.1 M phosphate buffer on ice. After washing, the specimens were post-fixed with 1% osmium tetroxide in 0.1 M phosphate buffer containing 1% potassium ferrocyanide and conductive-stained with 1% tannic acid solution and 1% osmium tetroxide solution. The specimens were dehydrated in graded series of ethanol and then critical point dried using a Samdri-PVT-3D system (Tousimis, Rockland, MD). The specimens were coated with osmium tetroxide using an osmium coater HPC-30 W (Vacuum Device, Ibaraki, Japan). Electron micrographs were captured with S-4800 field emission scanning electron microscope (Hitachi, Tokyo, Japan).