On day 6 after isolation, cell cultures of primary microglial cells were washed with warm PBS. Cells were fixed with 4% paraformaldehyde (PFA) in PBS for 10 min at 37 °C and subsequently blocked for 30 min with PBS containing 5% mouse serum. Cells were immunostained with P2RY12 (clone S16007D, Biolegend, 1:100) overnight at 4 °C, followed by staining with secondary goat-anti-rat-AlexaFluor594 antibody (clone Poly4054, Biolegend, 1:250), for 60 min. Staining of nuclei was performed with a DAPI-containing mounting medium (Roti®MountFluorCare, Carl Roth). Immunofluorescence images were acquired on a Keyence BZ-X800 fluorescence microscope.
