Anti fade fluorescence mounting medium

After perfusion with 4% paraformaldehyde, brains were removed, postfixed in the same solution at 4°C overnight, cryoprotected in 30% sucrose, and finally 30 μm coronal sections were obtained according to a previous report [29 (link)]. In immunofluorescence detection, antigen retrieval was performed by 70°C heating of the sections in sodium citrate buffer (10 mM trisodium citrate, 0.5% Tween-20 in H₂O, pH 6.0) for 30 min. The sections were blocked with 10% goat serum (with 0.5% Triton X-100) in Tris-buffered saline for 20 min and labeled with TH (Abcam, 1:2000), GFAP antibody (Abcam, 1:200), Iba-1 antibody (Woko, 1:200), or CD68 (Abcam, 1:200), in blocking buffer for 48 h at 4°C. After that, the slides were washed with PBS for three times (5 min each time), incubated with anti-rabbit secondary antibodies conjugated to Alexa Fluor 594 (Cell Signaling Technology, 1:1000) or Alexa Fluor 488 (Cell Signaling Technology, 1:1000) for 1 h. Thereafter, the slides were washed with PBS for three times again, and the sections were stained with DAPI. Finally, the sections were covered with coverslips and mounted with anti-fade fluorescence mounting medium (Beyotime Biotechnology) and observed under a fluorescence microscope (Model DMi8, Leica, Germany).

African green monkey renal epithelial (Vero) cells were purchased from Shanghai Cell Bank, Chinese Academy of Sciences (Shanghai, China). Dulbecco’s modified eagle medium (DMEM), fetal bovine serum, penicillin and streptomycin were obtained from HyClone Biochemical Products Co., Ltd. (UT, USA). Cell culture plates were purchased from Wuxi Nest Bio-Tech Co., Ltd. (Wuxi, China).
Rabbit anti-mouse IgG conjugated with fluorescein isothiocyanate (FITC-IgG), Lyso-Tracker Red, DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate DiIC18(3)), anti-fade fluorescence mounting medium were all purchased from Shanghai Beyotime Bio-Tech Co., Ltd. (Shanghai, China). Acridine orange (AO) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Sucrose, methyl-β-cyclodextrin and amiloride hydrochloride were purchased from Aladdin-reagent Co., Ltd. (Shanghai, China). Calcium chloride (CaCl₂), sodium oxalate (Na₂Ox), paraformaldehyde, ethanol were all analytical pure and purchased from Guangzhou Chemical Reagent Factory of China (Guangzhou, China).
X-L type environmental SEM (Philips, Eindhoven, Netherlands); confocal laser scanning microscope (LSM510 Meta Duo Scan, Zeiss, Germany); microplate reader (Safire2, Tecan, Männedorf, Switzerland); flow cytometer (FACS Aria, BD Corporation, Franklin Lakes, NJ, USA); fluorescence microscope (Leica DMRA2, Germany).