Leica em uc7 microtome

For transmission electron microscopy, whole mice were anesthetized with isoflurane using a VetEquip Vaporizer until a toe pinch yielded no response, and transcardially perfused via the left ventricle with 50 ml of phosphate-buffer saline (PBS) pH 7.5, followed by 30 ml of 2% glutaraldehyde (GA), 2% formaldehyde (FA), 2 mM calcium chloride in PBS. Cerebellum samples of 1 mm³ were dissected, washed three times in 0.1M cacodylate pH 7.4 and further fixed in 2% GA, 2% FA, 2 mM calcium chloride, 0.1M cacodylate pH 7.4 for 2 h at 4°C, washed four times for 10 min in the buffer and postfixed in 2% osmium tetroxide in the same buffer for 2 h on ice. The samples were washed twice in the buffer, five times in water, stained en bloc overnight in 2% aqueous uranyl acetate and washed three times in water, dehydrated in a series of ethanol concentrations and penetrated with EMbed 812 (EMS, Hatfield, PA), which was polymerized for 60 h at 65°C in flat molds. Thin (80 nm) sections of the samples were cut on a Leica EM UC7 microtome (Leica, Deerfield, IL) and stained with uranyl acetate/lead citrate. The samples were examined on a FEI Tecnai 20 electron microscope operated at 120 kV, and images were recorded on an AMT XR81 CCD camera.

Spermatozoa from AY078 and fertile controls were washed three times with 1× phosphate-buffered saline (PBS) at 2500 rpm at 25°C and then fixed with 2.5% glutaraldehyde (pH 6.9) for more than 2 h at 4°C.
For SEM, fixed samples were dehydrated using an ethanol gradient (30, 50, 70, 80, 90, and 100%; ×2), dried with a Quorum K850 Critical Point Dryer (Quorum Technology, Lewes, UK) after the ethanol had been replaced with hexamethyldisilamane, coated with a Cressington 108 Auto Sputter Carbon Coater (Cressington Scientific Instruments, Watford, UK), and observed using a ZEISS GeminiSEM 300 instrument (ZEISS, Oberkochen, Germany).
For TEM, fixed spermatozoa were post-fixed for 2 h at 4°C using 1% osmium tetroxide, dyed with 2% uranium acetate, dehydrated using a gradient, embedded in EPON 812 epoxy resin, cut into 100-nm sections using a Leica EM UC7 microtome (Leica, Wetzlar, Germany), stained with lead citrate, and examined using a Talos L120C G2 TEM (Thermo Fisher Scientific, Waltham, MA, USA).

This part references Małgorzata’s method [69 (link)] with some changes. One- to two-millimetre sections of the control and 10,000 mg/kg Pb (NO₃)₂-treated leaves, stems, and roots were fixed in 4% glutaraldehyde (v/v) in 0.2 mol/L sodium phosphate buffer (49 mL 0.2 mol/L Na₂HPO₄•12H₂O with 51 mL NaH₂PO₄•2H₂O in a total of 100 mL) at pH 6.8 at 4 °C for 12 h. The tissues were rinsed with 0.1 mol/L phosphate buffer (pH 6.8) six times (10 min each time) at room temperature, postfixed in 1% (v/w) OsO₄ at 4 °C for 2 h and then rinsed six times (10 min each time) in 0.1 mol/L phosphate buffer solution (pH 6.8). The samples were dehydrated using graded acetone and ethanol series (30, 50, 70, 80 and 90%) at room temperature, infiltrated, embedded in LR white resin and cut into ultrathin sections (~ 90 nm) using a Leica EM UC7 microtome (Leica, Nussloch, Germany). The sections were collected on copper grids, dusted with coal, and finally observed using a JEOL JEM-1230 transmission electron microscope (JEOL, Tokyo, Japan) at an accelerating voltage of 80 keV.