2.5. Immunofluorescence (IF) Analysis

Mice were anaesthetized and perfused with phosphate buffered saline (PBS). Then the brain tissues were obtained and fixed with 4% paraformaldehyde at 4 °C prior to the dehydration with 20% sucrose-PBS solution for 3 days and 30% sucrose-PBS solution for 3 days at 4 °C separately. The brain tissues were embedded with OCT glue and cut into continuous frozen slices with a thickness of 25 μm. Slices were permeabilized and blocked with 0.3% Triton X-100/5% BSA in PBS for 1.5 h at room temperature. Then the slices were incubated with NeuN (24307, Cell Signaling Technology, Danvers, MA, USA), Iba1 (17198, Cell Signaling Technology, Danvers, MA, USA), GFAP (MAB360, Millipore, MA, USA) or C3 (PA5-21349, Invitrogen, CA, USA) primary antibody at 4 °C overnight. After being washed with PBS, the second antibody anti-rabbit IgG (H + L), F(ab’)2 Fragment (Alexa Fluor^® 488 Conjugate) (4412, Cell Signaling Technology, Danvers, MA, USA) or anti-mouse IgG (H + L), F(ab’)2 Fragment (Alexa Fluor^® 555 Conjugate) (4409, Cell Signaling Technology, Danvers, MA, USA) was added and the slices were observed under a fluorescence microscope or a confocal microscope and analyzed using Image J software.

As to primary cell IF analysis, briefly, primary cells were rinsed with PBS, fixed with 4% paraformaldehyde at room temperature for 20 min, blocked with 0.3% Triton X-100/5% BSA in PBS for 1.5 h at room temperature and incubated with C3, microtubule-associated protein 2 (MAP2, 17490-1-AP, Proteintech, Chicago, IL, USA) or GFAP primary antibody. After being washed with PBS, the cells were incubated with anti-mouse IgG (H + L), F(ab’)2 Fragment (Alexa Fluor^® 555 Conjugate) or anti-rabbit IgG (H + L), F(ab’)2 Fragment (Alexa Fluor^® 488 Conjugate). To assess the average neurite length, 50 neurites of each field were measured and 5 fields of each group were counted. The neurite length was measured using Neuron J plug-in of Image J software. For C3 fluorescence intensity in GFAP-positive cell measurement, cells were observed under a fluorescence microscope and counted or analyzed using Image J software.