Immunofluorescence Staining of Microglia

Anesthetized mice were perfused transcardially with PBS, followed by 4 % paraformaldehyde (PFA) prepared in PBS. Tissues were post-fixed for at least 24 h in 4 % PFA, and transferred to 20 % sucrose solution before sectioning. Cryosections (40 μm) were permeabilized in 0.5 % PBS-Tween for 15 min and washed twice with PBS. After blocking in 5 % serum in PBS for 2 h at room temperature, sections were incubated overnight at 4 °C with antibody against Iba1 (ab5076, 1:400, Abcam), LRP1 (ab92544, 1:250, Abcam) and CD68 (14-0688, 1:50, eBioscience). After washing, sections were incubated with secondary antibodies conjugated to Alexa488 or Alexa647 (Life Technologies). Sections were mounted with Prolong Gold Anti-Fade Reagent containing DAPI (Life Technologies). All sections were imaged with a Leica TCS SP8 confocal microscope and analyzed with ImageJ. Sholl analysis was performed as described [24 (link)].