Tecnai spirit 120 kv tem

Samples to be examined were diluted into PBS (pH 7.4) to an appropriate concentration (~0.5 mg/mL) and dropped onto a freshly glow-discharged carbon-coated grid. After rinsing twice with PBS, the grid was stained with 1% phosphotungstic acid (pH 7.0) and then loaded onto a FEI Tecnai Spirit 120-kV TEM for examination.

The cells of each group were prefixed in 2.5% glutaraldehyde for 2 h and then cleaned 3 times in 0.1 M phosphate buffer (PB) for 60 s each time. Fixed at 4°C for another 1 h in a 1% osmic acid. Then, it was washed with 0.1 M PB for 3 times, stained in 1% uranyl acetate under 25°C for 1 h, and cleaned in ddH₂O for 3 times. The cells were progressively dehydrated in varying concentrations of ethanol solutions for 8 min, followed by cleaning in propylene oxide for 10 min. Later, the cells were coated with a compound of EPON812 resin and propylene oxide (2:1) under 4°C for 4 h, and then in pure EPON812 resin overnight. After roasting at 60°C for 24 h, the samples were cut into ultra-thin slices (70-nm thick) using the Ultracut UCT Microtome (Leica Biosystems, USA) and dyed in 1% uranium acetate for 20 min and lead citrate for 5 min. A Tecnai Spirit 120 kV TEM (FEI, USA) was applied to obtain the images.

Three-month-old C57BL6/J mice or twelve-month-old albino mice were transcardially perfused with cold fixative solution (2.5% glutaraldehyde and 2% PFA (paraformaldehyde) in 0.1 M phosphate buffer). Their eyes were removed, and the retinas were dissected and fragmented in 1 mm³ pieces. Retinal fragments were immersed in fixative solution (2.5% glutaraldehyde and 2% PFA in 0.1 M phosphate buffer) and incubated at 4 °C overnight. The retinal fragments were post-fixed in 1% osmium tetroxide 2% K₄Fe(CN)₆ in the dark for 2 h at 4 °C and rinsed in double-distilled water to remove the osmium. The retinal fragments were dehydrated in ascending concentrations of acetone, then infiltrated, and embedded in Epon (EMS). Blocks were obtained after polymerization at 60 °C for 48 h. Ultrathin sections of 60 nm in thickness were obtained using a UC6 ultramicrotome (Leica Microsystems, Vienna, Austria) and were stained with 2% uranyless and lead citrate. Sections were observed in a Tecnai Spirit 120 Kv TEM (FEI company, Eindhoven, The Netherlands), and images were acquired with a 1 k × 1 k CCD Megaview camera (Olympus Soft Imaging Solutions, Münster, Germany).