1400 plus tem

Fully differentiated cells in a six-well plate were quickly fixed with 2% glutaraldehyde in 0.1 M SC buffer (pH 7.4) at RT for 15 min then incubated at 4 °C for 15 min. Afterwards, cells were scraped down and pelleted by centrifugation. Cell pellets were postfixed in 1% OsO₄ in 0.1 M SC buffer for 1 h on ice. The cells were stained all at once with 2% uranyl acetate for 1 h on ice, then dehydrated in a graded series of ethanol (50–100%) while remaining on ice. The cells were then subjected to one wash with 100% ethanol and two washes with acetone (10 min each) and embedded with Durcupan. Sections were cut at 60 nm on a Leica UCT ultramicrotome and picked up on 300 mesh copper grids. Sections were post-stained with 2% uranyl acetate for 5 min and Sato’s lead stain for 1 min. Images were obtained using a Jeol 1400 plus TEM equipped with a Gatan digital camera.

Dissected adipose tissue was immediately fixed with 2–3 drops of fixative buffer (2% paraformaldehyde and 2.5% glutaraldehyde in 0.15 M sodium cacodylate buffer, pH 7.4). Fat tissues were gently removed and fixed at RT. After 2 h incubation, tissues were further cut into around 1-mm³ cubes and immersed in fixative buffer overnight at 4 °C. Tissue cubes were postfixed in 1% osmium 0.15 M sodium cacodylate (SC) buffer for 1–2 h on ice, followed by five 10-min washes in 0.15 M SC buffer, then rinsed in ddH₂O on ice. Washed tissues were stained with 2% uranyl acetate for 1–2 h at 4 °C then dehydrated in an ethanol series (50%, 70%, 90%, 100% and 100%, for 10 min each time) and dried in acetone for 15 min at RT. Dried tissues were infiltrated with 50:50% acetone:Durcupan for 1 h or longer at RT then changed to 100% Durcupan overnight. The next day, embedded tissues in Durcupan were placed in a 60 °C oven for 36 to 48 h. Ultrathin sections (60 nm) were cut on a Leica microtome with a Diamond knife and then post-stained with both uranyl acetate and lead. Images were obtained using a Jeol 1400 plus TEM equipped with a Gatan digital camera.