Ultra microtome uc6

Brains were perfused and preserved in 4% paraformaldehyde with 0.2% glutaraldehyde. Mediobasal hypothalamic brain tissue blocks of 1 mm³ were dissected and carefully washed in 0.1 M cacodylate washing buffer. Samples were post-fixed in 1% osmium tetroxide solution and subsequently incubated overnight in 1.5% uranyl acetate solution. Samples were extensively washed with Milli-Q water and then dehydrated with increasing concentrations of ethanol. Samples were then incubated in propylene oxide and later embedded in increasing concentrations of epon mix in propylene oxide. Finally, samples embedded in pure epon were placed in blocks for hardening at 60 °C. Ultrathin sectioning was conducted using an Ultra Microtome Leica UC6. Ultrathin sections of 70 nm were then collected in grids. Grids were contrasted with uranyl acetate and lead citrate before imaging. Images were obtained using a FEI Tecnai T12 transmission electron microscope at 100 kV. Microglia (identified by dense and highly heterochromatin nuclei22 (link)) and neurons that had contacts were imaged at × 11,000, × 23,000 and × 68,000 magnification for posterior analysis.

C2C12 cells were gathered from a cell culture dish and preserved in 5% glutaraldehyde, and diluted with phosphate buffer for at least 2 h. The cells were dissected into 1 mm³ and carefully washed in phosphate rinse solution for 15 min (three times). Cells were post‐fixed in 1% osmium tetroxide solution for 2–3 h and carefully washed in phosphate rinse solution for 15 min (three times), then dehydrated with increasing concentrations of ethanol. Cells were incubated in acetone and solidified in the oven. Ultrathin sectioning was then sliced by Ultra Microtome Leica UC6 in 70 nm and collected in grids. 3% uranyl acetate‐lead citrate double‐stained the grids. Images were obtained from a Jeol1230 transmission electron microscope at 120 kV at ×10,000, ×20,000, and ×50,000 magnification for posterior analysis.