Fei tecnai g2 spirit biotwin transmission electron microscope

Furthermore, we performed TEM in the granulosa cells of three PNA mice and three control mice to investigate autophagosome change in PNA mice’s ovaries. Ovaries from PNA mice and the control group were placed in a DMEM/F12 medium (Gibco BRL/Invitrogen, Carlsbad, CA, United States) containing 10% fetal bovine serum (HyClone, South Logan, UT, United States), 2 mM glutamine, 1 mM sodium pyruvate, 100 IU/ml penicillin, and 100 μg/ml streptomycin; 1-ml syringe needles were used to puncture the ovaries for releasing the granulosa cells, then the oocytes were filtered by a 40-μm cell strainer. The isolated granulosa cells were fixed with 2.5% glutaraldehyde for more than 6 h at 4°C and fixed in 1% osmium tetroxide in the above buffer for 1 h at room temperature. The samples were embedded in Epon after being dehydrated through a graded series of ethanol; 2% uranyl acetate and lead citrate were used for ultrathin sections staining. The TEM was performed using a FEI Tecnai G2 Spirit Biotwin transmission Electron Microscope (FEI Tecnai G2, America).

For electron microscopy studies, human spinal cords were fixed in 4% PFA overnight at 4 °C. After washing steps in 0.1 M PB, 200 µm coronal sections were cut on a Leica VT-1000 vibratome (Leica, Heidelberg, Germany). Sections were post-fixed with 2% osmium, rinsed, dehydrated, and embedded in Durcupan resin (Fluka, Sigma-Aldrich, St. Louis, USA). Semithin sections (1.5 µm) were cut with an Ultracut UC-6 (Leica microsystems, Wetzlar, Germany) and stained lightly with 1% toluidine blue. Finally, ultrathin sections (70–90 nm) were cut with a diamond knife, stained with lead citrate (Reynolds solution), and examined under an FEI Tecnai G2 Spirit BioTwin transmission electron microscope (ThermoFisher Scientific, Oregon, USA) using a Morada digital camera (Olympus Soft Image Solutions GmbH, Münster, Germany).

For cryo-electron microscopy experiments, lean and obese LNP samples at a concentration of ~10¹³ particles/ml were incubated with glow discharged carbon-coated copper grids (SPI supplies), following vitrification at 10 °C and 99% humidity by using a Leica EM GP automatic plunge freezer (Leica Microsystems Company). The excess sample was removed by blotting dry the grid for 2.5 s with filter paper and plunging it into liquid ethane at −180 °C. Following vitrification, grids were stored immersed in liquid nitrogen until use. Before imaging, the grids were mounted in a Gatan 626 cryo-holder (Gatan Company) and analyzed using an FEI Tecnai G2 Spirit BioTwin transmission electron microscope (Thermo Fisher Scientific). Pictures were taken with a Morada digital camera (Olympus Soft Image Solutions) and iTEM TIA image capture software (v4.7, Olympus).

Transmission electron microscopy was performed as described previously [30 (link)]. Hippocampal tissue (1 mm³) was fixed with 2.5% glutaraldehyde at 4 ℃ for 12 h, washed in 0.1 M cacodylate buffer three times, post-fixed in 1% osmium tetroxide for 2 h, and then dehydrated through a graded series of acetone solutions (30%, 50%, 70%, 80%, 90% and 100%). Tissues were cut into 50-nm sections using an ultrathin microtome (Leica, Wetzlar, Germany), contrasted with 2% uranyl acetate for 10 min and lead citrate for 5 min, and observed under an FEI Tecnai G2 Spirit Bio TWIN transmission electron microscope (Thermo Fisher Scientific).