Mab360

For immunofluorescence of brain slices, the sections were blocked with 5% FBS in PBST (0.3% Triton X-100 in PBS) for 1 h, followed by overnight incubation with primary antibody at 4°C. The sections were then washed with PBS and incubated with Alexa Fluor 488-conjugated goat anti-rabbit (1:1,000, Invitrogen, A11008) or Alexa Fluor 555 goat anti-mouse (1:1,000, Invitrogen, A21422) for 1 h at room temperature. The sections were ultimately rinsed with PBS and then mounted onto adhesive slides. Fluorescently labeled sections were visualized using an Olympus scanning microscope (Olympus BX51, Japan). For immunocytochemical staining, primary cells were rinsed with 0.1 M PBS and fixed with 4% paraformaldehyde for 20 min. Cell cultures on the cell slides were then prepared using the same procedures for the immunofluorescence of the brain slices.
The primary antibodies used for immunofluorescent staining were as follows: rabbit anti-TH antibody (1:1,000, abcam, ab6211), rabbit anti-GFAP antibody (1:1,000, abcam, ab7206), rabbit anti-Iba-1 antibody (1:1,000, Wako, 019-19741), rabbit anti-NeuN (1:100, CST, 24307), mouse anti-kir6.2 antibody (1:200, Santa Cruz, sc-390104), rabbit anti-C3 antibody (1:100, abcam, ab11887), mouse anti-GFAP antibody (1:1,000, Millipore, MAB360), mouse anti-Drp1 (1:200, Santa Cruz, sc-101270) and rabbit anti-TOM20 (1:200, Proteintech, 11802-1-AP).

GFAP and aldehyde dehydrogenase 1 family member L1 (ALDH1L1) expression in astrocytes were evaluated by indirect immunofluorescence. Astrocytes were fixed with 0.5 ml 4% paraformaldehyde in PBS for 30 min at 4 °C. After rinsing with PBS, cells were incubated with 10% normal goat serum in PBS with 0.1% Triton X-100 for 60 min. Then, slides were incubated overnight at 4 °C with mouse anti-GFAP antibody (1:400, Millipore Cat# MAB360, RRID:AB_11212597) or anti-ADLH1L1 (1:50, Santa Cruz Biotechnology, Cat# sc-100497, RRID:AB_2224180) in PBS with 0.1% Triton X-100 and 1% normal goat serum. After rinsing, slides were incubated for 1 h with goat anti-mouse Alexa 488 (1:800, Jackson ImmunoResearch Labs Cat# 115-545-003, RRID:AB_2338840). Slides were mounted with mounting medium containing DAPI (Abcam) and visualized in a fluorescence microscope Axiophot (Carl Zeiss, Germany). Negative control slides were incubated with normal goat serum instead of primary antibody. Acquisition was performed with a × 40 objective. Fluorescence was determined using ImageJ software and was normalized to the number of cells.