Talos120c transmission electron microscope

Cells were plated at 3 × 10⁶ and left overnight to adhere to the plate surface before transferring to hypoxia (0.1% O₂) for 24 h. Cells were fixed in 1 mL 0.1 M sodium cacodylate buffer (pH 7.4) containing 2.5% glutaraldehyde and 2% paraformaldehyde for 2 h and post-fixed with 1% osmium tetroxide and 1% potassium ferrocyanide for 1 h at 4°C, then block stained in 0.25% aqueous uranyl acetate, processed in a standard manner and embedded in Embed 812 (Electron Microscopy Sciences, Hatfield, PA.). Ultrathin sections (70 nm) were cut, mounted on copper grids and stained with uranyl acetate and lead citrate. Stained grids were examined under Talos120C transmission electron microscope (ThermoFisher Scientific, Hillsboro, OR) with Gatan (4k × 4k) OneView Camera. For morphometric analysis, mitochondria and cristae were identified manually from high-resolution, high magnification images acquired in a user-blinded fashion. Mitochondrial cristae length and maximal cristae width were measured using Fiji in 23 – 39 mitochondria per condition (Sood et al., 2014 (link)).

Purified human spermatozoa were collected and fixed with a freshly made fixative containing 3% paraformaldehyde and 0.1% glutaraldehyde in 0.1M phosphate buffer with 4% sucrose (pH 7.2). After washing with 0.1M phosphate buffer with 4% sucrose, 0.1M glycine was added to quench the unbonded aldehyde group. The cells were dehydrated in graded series of ethanol on ice and embedded in LR White (Electron Microscopy Sciences, Hatfield, PA). The samples were polymerized under UV light (360nm) at −10°C for 48 h, followed by 12 h at RT. The 90 nm-thin sections were cut and mounted on Formvar-Carbon coated 200 mesh nickel grids. The grid was incubated with an anti-TFAM antibody (1:1000, Abcam, #ab119684) in PBS with 1% BSA, and 0.05% Tween 20, for 2 h at RT, then overnight at 4°C. Upon washing, the anti-mouse secondary antibody conjugated with 18 nm gold (1:15, Colloidal Gold AffiniPure Goat Anti-Mouse IgG (H+L, EM Grade, Jackson ImmunoReasearch Laboratories, Inc.,) were added in PBS with 1% BSA, and 0.05% Tween 20, and incubated for 1h. After washing, the grids were stained with uranyl acetate and lead citrate by standard methods, imaged with Talos120C transmission electron microscope (Thermo Fisher Scientific), and recorded using Gatan (4k x 4k) OneView Camera with software Digital Micrograph (Gatan Inc).

lamC1−/− embryos were generated by in-crossing lamC1−/+ fish. 30 hpf embryos were fixed in EM fixative containing 2% paraformaldehyde, 2% glutaraldehyde in 0.1 M sodium cacodylate buffer at room temperature for 2 hrs and then overnight at 4°C. Fixed embryos were rinsed with 0.1 M sodium cacodylate buffer and post-fixed with 1% OsO₄ in 0.1 M cacodylate buffer, followed by block-staining with 1% uranyl acetate aqueous solution overnight at 4°C. The samples were rinsed with water, dehydrated in a graded series of ethanol, infiltrated with propylene oxide/Epon mixtures and finally embedded in EMbed812 (Electron Microscopy Sciences, PA USA). 70 nm sections were cut and mounted on 200 copper mesh grids and stained with uranyl acetate and lead citrate. Imaging was performed on a Talos120C transmission electron microscope (Thermo Fisher Scientific, Hillsboro, OR) with Gatan (4k x 4k) OneView Camera (Gatan, Inc., Pleasanton, CA). The primordium cells were pseudo-colored using Adobe Illustrator 2020 (Adobe).