Cultured neurons were labeled as previously described [37 (link), 48 (link)] with the following modifications. Depending on antibody vendors’ recommendations, cells were fixed with either freshly made 4% paraformaldehyde for 15 minutes at room temperature or with methanol for 15 minutes at − 20° C. After 3 washes with PBS, samples were blocked in PBS containing 5% normal goat serum and 0.25% Tween-20 for one hour. After blocking, samples were incubated with primary antibodies at 4° C overnight. The next day, samples were washed 3 times with PBS, and then incubated for 1 hour in Alexa Fluor®-tagged goat anti-mouse, anti-rabbit, or anti-chicken IgG secondary antibodies (ThermoFisher Scientific). For some experiments 4′,6-Diamidino-2-Phenylindole Dihydrochloride (DAPI; ThermoFisher Scientific) counterstaining was used between subsequent washes. Coverslips were then mounted onto microscope slides and allowed to dry overnight. Samples were then imaged using a Nikon Eclipse Ti inverted microscope equipped with a Yokogawa CSU-X1 spinning disk head, a 60x 1.4 NA Plan Apo objective; 405 nm, 488 nm, 561 nm and 640 nm lasers; and a Hamamatsu Flash 4.0 scientific CMOS camera. Analysis was performed using the Nikon software and ImageJ (https://imagej.nih.gov/ij/plugins/cell-counter.html ).
Brain tissue sections were labeled for immunohistochemistry as previously described[37 (link)].
Brain tissue sections were labeled for immunohistochemistry as previously described[37 (link)].