Cy3 conjugated goat anti rabbit immunoglobulin g igg

Brain slice preparation and imaging were completed according to the previously reported methods [5 (link),44 (link)]. The brains were soaked overnight in 4% paraformaldehyde solution. After dehydration was completed with 30% sucrose solution, the brain was sectioned with a thickness of 40 μm via microtome (Thermo Fisher Scientific, Waltham, MA, USA), collected in anti-freeze fluid, and stored at −20 °C for further use. For NeuN staining, sections were incubated with rabbit anti-NeuN (1:800, Abcam, Cambridge, MA, USA) primary antibody overnight at 4 °C. After washing 3 times with PBS, the slices were incubated with the secondary antibody Cy3-conjugated goat anti-rabbit immunoglobulin G (IgG) (1:400, The Jackson Laboratory, Bar Harbor, ME, USA) for 1 h at 37 °C. For GFAP staining, the primary antibody was goat anti-GFAP (1:800, Abcam, Cambridge, MA, USA) and the secondary antibody was rabbit anti-goat IgG conjugated with Cy3 (1:400, The Jackson Laboratory, Bar Harbor, ME, USA). After washing with PBS, all the brain slices attached to the microscope slides were counterstained with DAPI (1:4000, Beyotime, Shanghai, China) and sealed with 70% glycerol. Imaging was performed using a Leica TCS SP8 confocal microscope (Leica, Wetzlar, Germany) or an Olympus VS120 virtual microscopy slide scanning system (Olympus, Tokyo, Japan).

Brain slice preparation and imaging were completed according to the previously reported methods [22 (link),24 (link)]. The antibodies used were rabbit anti-NeuN (neuronal nuclei antibody; 1:800; Abcam, Cambridge, MA, USA), goat anti-GFAP (glial fibrillary acidic protein antibody; 1:800; Abcam, Cambridge, MA, USA), Cy3-conjugated goat anti-rabbit immunoglobulin G (IgG) (1:400; The Jackson Laboratory, Bar Harbor, ME, USA) and rabbit anti-goat IgG conjugated with Cy3 (1:400; The Jackson Laboratory, Bar Harbor, ME, USA). The sections were incubated with the primary antibody overnight at 4 °C. After washing 3 times with PBS, the slices were incubated with the secondary antibody for 1 h at 37 °C. After washing with PBS, all the brain slices attached to the microscope slides were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (1:4000; Beyotime, Shanghai, China) and sealed with 70% glycerol. Imaging was performed using an Olympus VS120 virtual microscopy slide scanning system (Olympus, Tokyo, Japan) or a Leica TCS SP8 confocal microscope (Leica, Wetzlar, Germany).

For staining neurons in vivo, the target brain slices were sealed with 10% sheep serum for 1.5 h, and immunostained with a Cy3-conjugated rabbit antibody against NeuN (diluted by PBS at 1:300, Merck Millipore, ABN78C3) overnight at 4 °C. For staining microglia cells in vivo, brain slices were blocked with 10% sheep serum in PBS with 0.3% Triton X-100 for 1.5 h, then incubated with primary antibody (anti-Iba1: diluted by PBS at 1:1000, LAK4357, WAKO) overnight at 4 °C. After washed 3 times with PBS, slices were incubated with the secondary antibody cy3-conjugated goat anti-rabbit immunoglobulin G (IgG) (1:400, 94,600, Jackson) for 1 h at 37 °C. All the brain slices above were washed 3 times with PBS, then attached to the microscope slides and sealed with 70% glycerol. Imaging was performed by using the Olympus VS120 Slide Scanner microscope (Olympus).