Cells and tissues were fixed with ice-cold methanol or 4% paraformaldehyde in PBS. After PFA fixation, permeabilization was effected with PBS containing 0.3% triton. Fixed and permeabilized cells were blocked with 3% normal donkey serum. Primary antibodies were against TREM2 (Abcam, 1:500), CD11b (Abcam,1:500), TMEM119 (Sigma/Atlas, 1:50), P2RY12 (Sigma/Atlas, 1:50), PU.1 (cell signaling, 1:500) and visualized by ad hoc secondary antibodies conjugated with Alexa 488, 568, 594, 647 (Life Technologies, 1:1000), followed by counter-staining with DAPI. Phase contrast and Fluorescent images of immuno-staining, as well as time lapse movies, were captured on a wide-field Nikon Ti2000 mounted with a SPOT RT monochrome camera. For 3D embedded microglia, observation was performed in the culture transwell, placed on a 1.5 coverslip Matek glass-bottom 35mm dish. Optical sectioning through 3D neural cultures was performed on a Zeiss LSM 700 at 20X magnification. Live imaging and wound assay in 3D culture of cells derived from iPS-wt5 was performed on Zeiss LSM710, photo-ablation was performed with 2-photon 780nm laser at 20% power, acquiring a 25um optical slice at 10X.
