(250–350 μl/slide) at appropriate dilutions (see below) and incubated overnight at room temperature. The next day, slides were washed with PBS-A solution 3 times for 5 min each. Tissue were blocked for 1 h using PBS-B solution at room temperature. After block, slides were incubated with Alexa Fluor® conjugated secondary antibodies (all at 1:500) and Hoechst stain (1X) in PBS-B (for 250–300 μl/slide) for 2 h at room temperature in the dark. After secondary staining, slides were washed 5 times with PBS for 5 min. Slides were cover slipped using fluoromount (Southern Biotech). For mouse brain IHC, brains were collected, fixed, and processed as mentioned above. Free-floating sections were blocked in blocking solution (1X PBS, 0.2% Triton X-100, and 10% goat serum) for 1 h at room temperature with gentle shaking. For human TMEM119 staining, heat mediated antigen retrieval was performed prior to blocking, as performed previously (Bennett et al., 2016). Free-floating tissue antigen retrieval was performed by placing floating sections in a 1.5 ml micro centrifuge tube containing 1 ml of Citrate Buffer solution and placing in a pre-heated temperature block set at 100°C. Tissue was heated for 10 min at 100°C then removed and allowed to come to
