Jem 1400 plus tem

Cells were fixed with freshly prepared fixative containing 2.5% glutaraldehyde in 0.15 M cacodylate buffer. Fixed cells were postfixed in 1% OsO₄ in 0.1 M cacodylate buffer for 1 hour on ice. Cells were stained en bloc with 2%–3% uranyl acetate for 1 hour on ice as described previously (95 (link)). The cells were dehydrated in a graded series of ethanol (20%–100%) on ice followed by 1 wash with 100% ethanol and 2 washes with acetone (15 minutes each) and embedded with Durcupan. Ultrathin (50–60 nm) sections were cut on a Leica UCT ultramicrotome, and picked up on Formvar and carbon-coated copper grids. Sections were stained with 2% uranyl acetate for 5 minutes and Sato’s lead stain for 1 minute. Grids were viewed using a JEOL JEM1400-plus TEM and photographed using a Gatan OneView digital camera with 4k × 4k resolution. Morphometric measurements were made randomly on deidentified samples. The free-hand tool in ImageJ was used to determine mitochondrial area by manually tracing around the mitochondrial outer membrane, as described previously (96 (link), 97 (link)). The area of each crista membrane was also calculated in the same manner. The sum of the areas of the total complement of cristae was then divided by the sum of the mitochondrial area to obtain the cristae density.

Three dpf fish were injected with 25 mg mL⁻¹ puromycin or vehicle. At 8 and 24 hpi, fish were fixed for TEM as described by Lyons et al.^{43 (link)} Briefly, fish were fixed in modified Karnovsky's solution (2% glutaraldehyde and 4% paraformaldehyde in 0.1 M sodium cacodylate buffer), postfixed in 2% osmium tetroxide in 0.1 M imidazole and 0.1 M sodium cacodylate, stained en bloc in saturated uranyl acetate, and dehydrated into 100% acetone. All of these steps were accelerated using microwave stimulation with a Panasonic microwave with inverter technology. Temperature throughout was maintained at 15°C in a cooled water bath. Following dehydration, embryos were embedded in Epon. Ultrathin sections (60 nm thick) were cut from selected areas and mounted on Formvar/carbon-coated copper slot grids. After drying, these were stained in 1% aqueous uranyl acetate and Reynold's lead citrate, and then viewed in a JEOL JEM-1400 Plus TEM.
Representative images were collected on a GATAN OneView camera for analysis of GFB structural changes using Fiji, as similarly performed by Benchimol de Souza et al.⁴⁴ The mean podocyte frequency per μm length (of basement membrane) and mean podocyte width were determined in all clearly identifiable sections of the GFB (Fig. 3A).

For visualizing the shape and contour of the HP1γ dimer, we produced and purified an N-terminal 6×His-tagged recombinant form of this protein using the pET vector system (Novagen, CA). The HP1γ-encoding plasmid was grown in DE3 BL21 bacteria cells overnight and induced with 0.5 mM IPTG for 90 min at 32 °C. The recombinant protein was purified using the Thermo Scientific HisPur Cobalt Resin Kit according to the manufacturer’s instructions. Protein was dialyzed overnight and concentrated to a final concentration of 1 mg/ml. For visualization at the electron microscopy level, 10 μl of the purified protein solution was placed on the surface of glow-discharged formvar carbon-coated grids. After 30 s, the grids were blotted and stained for 30 s in 1 % uranyl acetate. Micrographs were acquired using a JEOL, JEM-1400Plus TEM at 80-kV accelerating voltage, equipped with a Gatan Orius 832 camera.