Transmission electron microscopy (TEM)

Mice were put down by isofluran and perfused trans-cardiacally for 2 mins with PBS followed by perfusion for 8 mins with freshly prepared 4% PFA + 1% glutaraldehyde buffer under 120 mm Hg pressure. Brain tissues were immersed in cacodylate buffer and cut at 400 μm thick sections using a Vibratome (Leica, VT1000S). After washing three times with cacodylate buffer, a post fixation with 1 % osmium tetroxide in cacodylate buffer was done according to previous methods⁶². 0.5 μm² sections of areas of interest were selected. 80 nm ultra-thin sections were cut and stained with lead citrate and examined using an EM900 (Zeiss) TEM.