Tecnai 12 120 kv transmission electron microscope

Immediately after removing the testis and EDs from the right side, each mouse was fixed by perfusion via the heart’s left ventricle with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4 containing 0.05% calcium chloride. After 10 min of perfusion, the left testis, efferent ducts, and epididymis were excised and cut into small 1 mm³ blocks, with samples taken of each of the 4 epididymal regions. All samples were placed in fresh fixative on ice for several hours and then washed overnight at 4°C in 0.1 M sodium cacodylate buffer (pH 7.4). The next day, samples were washed three times for 10 min each in cacodylate buffer and immersed for 2 hours in a solution containing 1% osmium tetroxide and 1.5% potassium ferrocyanide. All tissues were dehydrated in a graded series of acetone and embedded in Epon 812 medium (Mecalab Ltd., Montreal, QC). Thin sections (60 nm) of the testis, efferent ducts and 4 regions of the epididymis were cut with a diamond knife and mounted on copper grids for EM analysis and stained for 5 min with uranyl acetate and 3 min with lead citrate; grids were examined with a FEI Tecnai 12 120 kV Transmission Electron Microscope (TEM). Epon blocks of these tissues were also cut with glass knives (5 μm), placed on glass slides and stained with toluidine blue for LM analysis.

Gut lavages and vesicles were coated directly on formvar carbon grids (Agar Scientific Ltd, Stansted, Essex, UK). Gut lavages were fixed with 1% glutaraldehyde in 0.1 M sodium cacodylate buffer for 1 minute and stained with 1% uranyl acetate for 1 minute. Isolated vesicles were fixed with 2% glutaraldehyde (Millopore Sigma, Burlington, MA, USA) washed three times with autoclaved Milli-Q water and stained with 2% uranyl acetate for 1 minute each. For ultrastructure analysis, dissected midguts from infected or uninfected sandflies were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer overnight. After washes in the same buffer, samples were post-fixed with osmium tetroxide and dehydrated in a graded acetone series prior to embedding in epoxy resin. Ultrathin sections (70–80 nm) were cut from resin blocks using a Reichert-Jung Ultracut E Ultramicrotome (Biel, Switzerland). Formvar grids covered with gut lavages or with ultrathin sections, as described above, were visualized in the FEI Tecnai 12 120 kV transmission electron microscope. Images were taken with the AMT XR-80C CCD Camera System. Intraperitoneal vesicle samples were visualized with the FEI Tecnai G² Spirit Twin 120kV Cryo-TEM and images were taken with the Gatan Ultrascan 4000 4kx4k CCD Camera System, model 895 (Facility for Electron Microscopy Research, McGill University, QC, Canada).