Tecnai g2 spirit transmission electron microscope

Stag2^f/y and Stag2^f/y;Nes^Cre P18 pups were transcardially perfused with 4% paraformaldehyde, 1% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4). Tissues were dissected and fixed with 2.5% (vol/vol) glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for at least 2 hr. After three rinses with the 0.1 M sodium cacodylate buffer, optic nerve samples were embedded in 3% agarose and sliced into small blocks. All samples were again rinsed with the 0.1 M sodium cacodylate buffer three times and postfixed with 1% osmium tetroxide and 0.8% potassium ferricyanide in the 0.1 M sodium cacodylate buffer for 3 hr at room temperature. Blocks were rinsed with water and en bloc stained with 4% uranyl acetate in 50% ethanol for 2 hr. Samples were dehydrated with increasing concentrations of ethanol, transitioned into propylene oxide, infiltrated with Embed-812 resin, and polymerized in a 60°C oven overnight. Blocks were sectioned with a diamond knife (Diatome) on a Leica Ultracut 7 ultramicrotome (Leica Microsystems) and collected onto copper grids, poststained with 2% aqueous uranyl acetate and lead citrate. Images were acquired on a Tecnai G2 Spirit transmission electron microscope (Thermo Fisher) equipped with a LaB6 source using a voltage of 120 kV. Tissue processing, sectioning, and staining were completed by the Electron Microscopy Core at UT Southwestern Medical Center.

The transmission electron microscopy (TEM) images were acquired by a Tecnai G2 Spirit Transmission Electron Microscope (Thermo Fisher Scientific, Waltham, MA, USA) with an acceleration voltage of 120 kV. All the cell samples were first resuspended using 20 μL double‐distilled water (ddH₂O) and then centrifuged at 800g, 24°C for 10 seconds. Five microliters (5 μL) of supernatant was applied to coat the carbon‐coated copper grid (300 meshes; Beijing Zhongjingkeyi Technology Co., Ltd., Beijing, China) for 60 seconds. The grid was washed twice using 5 μL ddH₂O for 45 seconds then 5 μL of 2% uranyl acetate was applied for 60 seconds. In each step, the solution was blotted away using filter paper touching the edges of the grids.