Anti neun antibody

Immunohistochemical staining was performed as described previously^{44 (link)}. Briefly, mouse brains were transcardially perfused and subsequently fixed with 4% paraformaldehyde overnight. The brain tissues were sectioned through the region of interest at a thickness of 20–30 μm, and endogenous peroxidase activity was blocked with 3% H₂O₂. Thereafter, sections were incubated with anti–CD11b antibody (1:4000, Abcam, Cambridge, UK) at 4 °C overnight, rinsed three times with PBS, and incubated with the secondary antibody at 25 ± 1 °C for 1 h. For fluorescent staining, sections were blocked with 3% bovine serum albumin (BSA) for 30 min, and subsequently incubated with anti-Iba-1 antibody (1:100; Abcam), and anti-NeuN antibody (1:100; Abcam) at 4 °C overnight. Thereafter, sections were rinsed thrice in PBS and incubated with fluorescent-labeled secondary antibodies at 25 ± 1 °C for 1 h in the dark. Samples were visualized using Olympus BX53 microscope (Olympus, Tokyo, Japan), and the images analyzed using Image J (NIH, Bethesda, MD, USA).

For immunofluorescence staining of frozen tissue sections and primary cultured neurons, all sample incubation solutions were prepared using phosphate-buffered saline (PBS) supplemented with 10% goat serum and 0.1% Triton X-100. The rabbit polyclonal anti-p8 antibody (1:200, Santa Cruz, Dallas, TX, USA) and the fluorescein (FITC)-conjugated goat anti-rabbit IgG antibody (1:50, DingGuo, Dalian, China) were used together with (4′,6′-diamidino-2-phenylindole, DAPI) for nuclear labeling. We used anti-NeuN antibody (1:1000, rabbit, Abcam, Cambridge, UK) as neuronal marker to detect neurons (data not shown). The frozen tissue section samples were incubated with blocking buffer for 30 min at room temperature and then with primary antibody at 4°C overnight. After washing with PBS for three times, the samples were incubated with secondary antibody for 1 h at room temperature. Microphotographs were taken using a fluorescence microscopy (A1+/A1R+, Nikon, Tokyo, Japan). All digital images were processed using the same settings to improve the contrast.

Mouse brain sections were incubated with anti-mouse transferrin (primary antibody; Bethyl) followed by anti-goat IgG-HRP (secondary antibody; Jackson Immuno Research Inc., West Grove, PA, USA). Human brain sections were incubated with anti-human transferrin antibody (DakoCytomation, Glosyrup, Denmark) followed by HRP-conjugated secondary antibody (ab205718, abcam). Signals were developed with ImmPACT DAB substrate (Vector Lab.). For double staining, DAB-stained slides were treated with quenching peroxidase and then with anti-NeuN antibody (abcam). The sections were further incubated with anti-rabbit IgG-HRP (abcam). Signals were developed with VECTOR VIP substrate (Vector Lab.).

After the end of administration, the freshly dissected brain tissue (7 μm) was fixed in formalin and embedded in paraffin for preparation of sections. The sections were then deparaffinized and rehydrated before being repaired in citrate buffer (pH 9.0) at the temperature of 80°C for 50 min. Then 3% hydrogen peroxide was used to quench endogenous peroxidase before being blocked with 10% goat serum in an incubator (37°C) for 30 min. Thereafter, the sections were incubated with anti-NeuN antibody (1:100, Abcam, MA, USA) at the temperature of 4°C for 48 h. The secondary incubation was conducted with goat anti-mouse DyLight 488 antibody (1:200, Abcam, MA, USA) at room temperature (37°C) for 2 h. The next step was DAPI staining, which is followed by quenching of fluorescence and mounting. After that, morphology of sections was observed under a laser scanning confocal fluorescence microscope and photographed at the indicated sites. Quantified analysis was performed to positive cells present in the sections at lens of 20 × and 40 ×, respectively, and the number of NeuN-positive cells was calculated in a random microscope field of each slice by CellF software.