Az 100 multipurpose microscope

Brain processing and immunostaining were performed on free-floating sections. Mice were anesthetized with 2,2,2-tribromoethanol (350 mg/kg, Sigma-Aldrich, T48402) and transcardially perfused with 0.9% saline, and brains were removed and fixed in phosphate-buffered 4% paraformaldehyde for 48 h before cryoprotection with 30% sucrose. Mouse brain sections (30 μm) or fixed cultured cells were washed three times with PBS, and antigen retrieval was performed using citrate buffer (pH 7.0); samples were then permeabilized and blocked in PBS containing 0.5% Triton X-100 and 10% normal goat serum at room temperature for 1 h. Sections were incubated with primary antibodies in blocking buffer overnight at 4 °C. After washing, secondary antibodies were added to the blocking buffer and incubated for 1 h. Samples were then washed and counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Images were acquired under a fluorescence microscope (Nikon AZ-100 multipurpose microscope). Primary antibodies used for immunostaining include GFAP (mouse, 1:500; Abcam, ab10062), IBA1 (Rabbit, 1:300; Abcam, ab178847), and MAP2 (goat, 1:500; Abcam, ab32454). Donkey anti-mouse/rabbit 488/594 secondary antibodies (1:1000) and mounting medium with DAPI were purchased from Invitrogen.

For immunofluorescence, cells were fixed in 4% formaldehyde for 20 min at room temperature, permeabilized with 0.3% Triton X-100 in PBS for 10 min, and then incubated with 2% BSA and 0.05% sodium azide in PBS for 1 h at room temperature to block nonspecific antibody binding. Subsequently, cells were incubated with primary antibodies against the cell markers of interest overnight at 4°C. Cells were then washed three times for 10 min with PBS and incubated for 1 h at room temperature with an Alexa Fluor 594-conjugated secondary antibody (Invitrogen). Then, the cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) in Dulbecco's PBS (DPBS; Sigma-Aldrich) and analyzed under a fluorescence microscope (Nikon AZ-100 multipurpose microscope).