Immunofluorescent Staining of Microglia

Cells were subjected to a 5 min wash with 1× phosphate buffered saline (PBS) and fixed for 20 min with PBS containing 4% (w/v) paraformaldehyde and 4% (w/v) sucrose, pH 7.4 at room temperature. Incubation for 5 min with PBS containing 0.1% (v/v) Triton X-100 was done to permeabilize cells. Microglia were subsequently blocked for 1 h with PBS containing 10% (v/v) goat serum and incubated overnight at 4°C with rabbit Iba-1 (1:750; Wako) in blocking buffer containing 10% goat serum. After incubation with Alexa Fluor 546 goat IgG secondary antibody (1:1000) in PBS containing 0.1% (v/v) triton X-100 and 1% goat serum, cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; 13.0 ng/μL) in PBS, followed by two 5 min PBS washes (Daniele et al., 2015 (link); Sánchez and Maguire-Zeiss, 2020 (link)). Coverslips were subsequently mounted with Hydromount (Electron Microscopy Services) in preparation for imaging with the Nikon Eclipse Ti-S inverted fluorescent microscope.