Chronic hM3Dq signaling in microglia ameliorates neuroinflammation in male mice

William Binning; Aja E. Hogan-Cann; Diana Yae Sakae; Matthew Maksoud; Valeriy Ostapchenko; Mohammed Al-Onaizi; Sara Matovic; Wei-Yang Lu; Marco A. M. Prado; Wataru Inoue; Vania F. Prado

CX3CR1CreER-hM3Dq mice underwent intracardiac perfusion with 1X PBS for 5 min and 4% PFA in PBS for 5 min. Brains were extracted and postfixed in 4% PFA at 4°C for 24 h. Brains were then cut into 40-µm thick sections using a vibratome (VT1200S, Leica). Staining for Iba1, HA, NeuN or GFAP was performed as described previously (Kolisnyk et al., 2016) using Alexa Fluor 555/633tagged secondary antibodies and Hoechst. Sections were mounted on glass slides using Fluorescent-G mounting medium (Electron Microscopy Sciences, cat#17984-25) and imaged using an SP8 confocal microscope (Leica) equipped with a 20x/0.75 dry or a 40x/1.3 oil objective. At least 4 sections were imaged for each animal (N=5) for each condition, with at least 6 fields imaged for each section. Images were analyzed using ImageJ Cell Counter plugin. 075-CL) containing 10% heat-inactivated fetal bovine serum (Gibco, cat#10438026) and 1× penicillin/streptomycin (Gibco, cat#15140-122) . Cells were plated on 75-cm2 tissue culture flasks coated with poly-D-lysine (10µg/mL, Sigma Aldrich, cat#P9155) and incubated at 37 °C in 5% CO2 atmosphere. The culture medium was changed after 3 days and then every 5 days until confluency (12-18 days in vitro).