Fluoview fvx confocal scan head

Tissue sections (10 μm) were incubated in normal goat serum (Zhongshan Golden Bridge, Beijing, China) for 30 min followed by incubation with a mixture containing a PDE10A antibody (rabbit polyclonal antibody, 1:100), a microtubule-associated protein 2 (MAP2) antibody (chicken polyclonal antibody, 1:100, Zhongshan Golden Bridge) and a glial fibrillary acidic protein (GFAP) antibody (mouse polyclonal antibody, 1:100, Zhongshan Golden Bridge) overnight at 4°C. Sections were washed twice in PBS and incubated with a mixture of DyLight 488-conjugated goat anti-rabbit IgG (1:200, Zhongshan Golden Bridge), DyLight 549-conjugated goat anti-mouse IgG (1:200, Zhongshan Golden Bridge) and DyLight 405-conjugated goat anti-chicken IgG (1:200, Zhongshan Golden Bridge) in a darkroom for 90 min at 37°C. Tissue sections were mounted in 50% glycerol/PBS. Fluorescence was detected using laser scanning confocal microscopy (Leica Microsystems Heidelberg GmbH, Germany) on an Olympus IX 70 inverted microscope (Olympus, Japan) equipped with a Fluoview FVX confocal scan head. To determine the specificity of antibodies, PDE10A antibody (488 channel), GFAP antibody (549 channel) and microtubule-associated protein 2 (MAP2) antibody (405 channel) were replaced by PBS with the same protocols.

The rat cryosections were thawed at room temperature and then fixed in acetone solution for 30 min before staining. After several washes in PBS, the sections were preincubated with 0.4% Triton for 30 min, washed in PBS, and blocked with normal goat serum (Zhongshan Golden Bridge, Beijing, China) at room temperature for 1 h. For double labeling, the tissue sections were incubated with a mixture of anti-Npas4 antibody (1∶100, Novus biological, USA) and anti-MAP2 antibody (1∶100, mouse monoclnal antibody, Boster, Wuhan, China) or mouse anti-GFAP antibody (1∶100, Zhongshan Golden Bridge, Beijing, China) at 4°C overnight. Tissue sections were washed with PBS and incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (1∶50; Zhongshan Golden Bridge, Beijing, China) and Tetramethyl rhodamine isothiocyanate(TRITC)-conjugated goat anti-mouse IgG (1∶50; Zhongshan Golden Bridge, Beijing, China) in the dark room for 90 min at 37°C, then washed with PBS three times for 10 min each, and mounted in 1∶1 glycerol/PBS. Fluorescent images were collected using laser scanning Confocal microscopy (Leica Microsys-tems Heidelberg GmbH, Germany) on an Olympus IX 70 inverted microscope (Olympus, Japan) equipped with a Fluoview FVX confocal scan head.

IF was conducted as previously described,³⁶ (link) with some modifications. The frozen sections of the samples from different groups were incubated in normal goat serum and then with a mixture containing a TNIK antibody (sc-377215, mouse polyclonal antibody, 1:100), a glial fibrillary acidic protein (GFAP) antibody (rabbit polyclonal antibody, 1:100, Proteintech), and a microtubule-associated protein 2 (MAP2) antibody (chicken polyclonal antibody, 1:1000, GeneTex) overnight at 4 °C. Sections were washed and incubated with a mixture of DyLight 488-conjugated goat anti-mouse IgG (1:1000, Abcam), DyLight 405-conjugated goat anti-rabbit IgG (1:1000, Abcam), and DyLight 555-conjugated goat anti-chicken IgG (1:1000, Abcam) in a darkroom for 120 min at 37 °C. After washing, the sections were mounted with 80% glycerol. Fluorescence was detected using laser scanning confocal microscopy (Leica Microsystems Heidelberg GmbH, Germany) on an Olympus IX 70 inverted microscope (Olympus, Japan) equipped with a Fluoview FVX confocal scan head.