Ex 2 transmission electron microscope

Mice were perfused with 4 % PFA, brains were removed and immersion fixed. Brain sections were cut using a vibratome (100 μm) and incubated in 0.5 % gelatin, 5 % horse serum and 0.01 % saponin in PBS for 5 h. Microglia staining was performed using a Rb anti-Iba1 (1:600; Wako), a secondary biotinylated anti-Rb (1:500; Rockland), streptavidin-HRP (1:1000; Rockland, S000-03) for 3 h and visualized using DAB Substrate Kit (Cell Marque, 957D). Tissue was then fixed with 2 % PFA, 2.5 % GA and sections were processed using routine EM protocols. Grids were not counterstained to preserve immunostaining. Finished blocks were sectioned using a DiATOME ultra 45° diamond knife and a LEICA Ultracut UC7. Sections (90 nm) were cut and picked up onto 200 hex mesh, formvar-carbon coated copper grids (Ted Pella, 01800-F). Images were captured using a JEOL 1200 EX II Transmission Electron Microscope with AMT digital camera.