Rabbit anti neun

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).

After the behavioural tests, the mice were anaesthetized and transcardially perfused with saline. Then, the brains were removed, and the hemispheres were separated. Brain samples assigned for immunohistochemical staining were immersed in OCT at -80 °C before coronal sectioning on a Frozen slicer (Thermo Fisher Scientific, USA) (10 μm thickness). The sections were restored to room temperature, fixed with cold acetone for 5 min, and permeabilizing agent was used for 5 min. Then, the cells were rinsed three times at room temperature for 5 min each with 1x PBS (pH 7.2-7.4) and blocked with 10% bovine serum in 0.1% Triton X-100 for 30 min. The blocking solution covered all brain tissues and prevented the tissue from drying out. Then, the samples were incubated with primary antibodies overnight at 4 °C. The primary antibodies included rabbit anti-NeuN (1:500, Abcam), rabbit anti-GFAP (1:200, CST), rabbit anti-Iba-1 (1:100, Abcam), rabbit anti-CD68 (1:300, Abcam), rabbit anti-APP (1:100, Millipore), and mouse anti-β-amyloid specific for Aβ₄₂ (1:200, CST). The next morning, the sections were incubated for 1 h at room temperature with DyLight 488-/555-conjugated goat anti-rabbit/mouse IgG (1:200, Abcam) and stained with DAPI for 3 min. Fluorescence images were captured using an Olympus FV3000 confocal microscope.

For immunodetection of Glutamatergic neurons, the sections were blocked in the solution (containing 0.3% Triton X-100, 0.25% BSA (bovine serum albumin) and 5% GS (goat serum)) for 2 h at RT. Then the sections were incubated with primary antibodies (rabbit anti-vGluT1, 1:1000, Abcam; rabbit anti-NeuN, 1:3000, Abcam) overnight at 4 °C and with fluorescent secondary antibody (at 1:1000, Abcam) at RT for 90 min. After DAPI incubation for 5 min, they were mounted by an anti-fluorescence quencher. Finally, images of the sections were taken with a confocal fluorescence microscope (Leica) and fluorescence intensity was quantified with the Image-Pro plus software. Image J software was used to digitize the fluorescence images and the figures were converted into 8-bit gray images. Then the selected area was analyzed to determine its surface area and the average, minimum, and maximum gray values and integrated optical density (IOD) were calculated. All images were set to the same light intensity and exposure. The sample size for each group was n = 5.

Free-floating sections were used for immunofluorescence labeling. For general immunofluorescence staining, all sections were washed three times with 0.01 M PBS and blocked for 30 min at 37°C by 0.3% Triton X-100 with 5% fetal bovine serum (HyClone, Logan, Utah). Next, sections were incubated with primary antibodies overnight at 4°C and then tagged with Cy3-conjugated second antibody (1:200, Beyotime, Shanghai, China) at 37°C for 30 min. The nuclei were visualized by DAPI (Beyotime) staining after immunofluorescence labeling. Finally, the sections were mounted on the slides and covered with cover slides for the confocal microscope. For BrdU staining, the DNA denaturation step of 2 N HCl (30 min, 37°C water bath) was added before blocking, so that BrdU could be incorporated into the DNA of mitotic cells.
The main antibodies used in this study were as follows: mouse anti-BrdU (1:200, Abcam) for proliferating cells, rabbit anti-doublecortin (anti-DCX; 1:200, CST, Boston, MA, USA), mouse anti-GFAP (1:200, CST) for astrocyte, and rabbit anti-NeuN (1:200, Abcam, Cambridge, ENG) for mature cells. For secondary antibodies, Cy3 conjugate (1:200, Beyotime) antibodies were used. The images were captured on Nikon A1R + laser scanning confocal microscope (Nikon, Tokyo, JP). Quantification and image analysis referred to the previous description (Zeng et al., 2016 (link)).

Immunohistochemistry and immunoblotting were performed as previously described 4 (link), 34 (link). The following antibodies for immunohistochemistry were used: rabbit anti-GFP (#A6455, Invitrogen), rabbit anti-MCP-1 (#500-P113, PeproTech), mouse anti-TNFα (#ab8348, Abcam), rabbit anti-NeuN (#ab177487, Abcam), rabbit anti-Iba1 (#019-19741, Wako), rabbit anti-Ki67 (#ab15580, Abcam), rat anti-CD68 (#FA-11, Abcam), rabbit anti-Tmem119 (#ab209064, Abcam), rat anti-P2RY12 (#848001, BioLegend), rabbit anti-Synaptotagmin 1 (#105003, Synaptic Systems), mouse anti-MHC-II (#ab55152, Abcam), and mouse anti-PSD-95 (#MAB1596, EMD Millipore). Species-specific secondary antibodies were conjugated with Alexa Fluor488, 594 or 647 (Molecular Probes, Invitrogen). The antibodies for immunoblotting were: rabbit anti-iNOS (#610332, BD PharMingen), mouse anti-COX2 (#610204, BD PharMingen), and mouse anti-GAPDH (#MAB374, EMD Millipore).

Cells were cultured on a round slide. After treatment, the slide was washed with 0.01 M PBS and fixed with 4% PFA as previously described (34 (link)). The primary antibody (Mouse anti-α-synuclein: BD, USA; Rabbit anti-NeuN: Abcam, USA) was added and incubated with the cells for 48 h at 4°C. Then, the slide was exposed to the Alexa Fluor^® antibody (Invitrogen, USA) and incubated for 1 h at 37°C. The nucleic acids were stained with DAPI (Invitrogen, USA). Following a final wash and mounting with anti-fade medium (Sigma, USA), images were acquired using a fluorescence microscope (Nikon, Japan). The fluorescence intensity was determined using Image-Pro Plus, Version 6.0 (MediaCybernetics, Inc., USA).

In order to analyze the specificity of the adenovirus in vivo, the animals were injected with the adenovirus, and the brains were collected at 48 and 96 h. The rat brains were fixed in 4% PFA by immersion for 48 h. Free-floating frontal hypothalamic slices of 40 μm thickness were obtained by a cryostat and subsequently processed. Tissues were stained with chicken anti-vimentin (1:200; Millipore, Billerica, MA, USA), mouse anti-GFAP (1:200; Millipore), and rabbit anti-NeuN (1:5000; Abcam, Cambridge, MA, USA) antibody diluted in Tris-HCl buffer (pH 7.8) containing 1% bovine serum albumin. After extensive rinsing, the sections were incubated for 2 h at room temperature with Cy2- or Cy3-labeled secondary antibodies (1:200; Jackson ImmunoResearch Laboratories). TOPRO-3 (1:1000; Invitrogen) was used as nuclei staining. The slides were visualized and captured using confocal laser microscopy LSM 700 (Zeiss).