Anti gfap

Anti-Trk A and anti-Myc tag antibodies were purchased from Cell Signaling Inc. Anti-nestin, anti-Sox2 and anti-GFAP were purchased from Merck. Anti-Ki67, anti-Sox9 and anti-doublecortin were purchased from abcam. Anti-neurogenin 2 was purchased from R&D. Anti-NgR1 and anti-beta III tubulin were purchased from Sigma. Anti-phospho NgR1 (serine²⁸¹) primary rabbit antibody was prepared by Cambridge Research Biochemicals Limited (Cambridge, UK).

NSCs that were cultured on coverslips were fixed with 4.5% PFA in PBS, washed with PBS, blocked in 5% donkey serum (Merck Life Science, catalog no. S30-100ML) in PBST (PBS  +  0.2% Triton X-100, VWR, catalog no. 0694-1 L), and incubated overnight with primary antibodies (anti‐TCF7L2, Cell Signaling Technology, catalog no. 2569, 1:250 dilution; anti-SOX9, Biotechne, catalog no. AF3075-SP, 1:250 dilution; anti-GFAP, Merck Life Science, catalog no. Hpa056030, 1:250 dilution). The next day, the coverslips were washed and incubated with Alexa Fluor‐conjugated secondary antibodies (donkey anti-rabbit immunoglobulin G [IgG] with Alexa Fluor 488, Invitrogen, catalog no. A‐21206, 1:500 dilution; donkey anti-mouse IgG with Alexa Fluor 594, Invitrogen, catalog no. A‐21203, 1:500 dilution; donkey anti-mouse Author: IgG with Alexa Fluor 555, Invitrogen, catalog no. A-31570, 1:500 dilution; donkey anti-Goat IgG with DyLight 650, Invitrogen, catalog no. SA5-10089, 1:500 dilution). Finally, the coverslips were mounted on microscope slides with Fluoromount-G Mounting Medium with DAPI (Invitrogen, catalog no. 00-4959-52). Images were captured with an Axio Imager Z2 LSM 700 Zeiss confocal microscope.

Slides were washed in PBSX1 and PBS-TWEEN 20 0.5% (Sigma-Aldrich, St. Louis, MO, USA). After incubation in BSA, slides were incubated overnight at 4 °C with the following primary antibodies: anti-GFAP (1:200,04-1062, rabbit anti-mouse; Merck Millipore, Merck KgaA, Darmstadt, Germany) or anti-vimentin (1:500, AB-5733, chicken anti-mouse; Merck Millipore). Sections were washed and incubated for 1 h at room temperature with the following secondary antibodies: goat anti-rabbit IgG Alexa Fluor 488 (1:200, A11008, Molecular Probes, Invitrogen, Carlsbad, CA, USA) and goat anti-chicken IgG NL-557 (1:200,NL016; R&D Systems-Biotest, Minneapolis, MN, USA). They were then stained with NucBlue (#R37605; Molecular Probes, Eugene, OR, USA) and covered with ProLong Gold Antifade Mountant (#P36930; Molecular Probes). Slides were shielded from light and maintained at 4 °C. Images were obtained using a fluorescence microscope equipped with a DAPI filter (ApoTome; Carl Zeiss Microscopy, Jena, Germany).

Fifty male Wistar rats (280–300 g) were obtained from shanghai laboratory animal center, Chinese Academy of Sciences. Four rats were kept in a cage and allowed free access to water and food. The house was maintained at 22–25°C with a 12-hr light-dark cycle.
Mouse monoclonal anti-PSD-95 (postsynaptic density protein-95) and anti-synaptophysin were respectively obtained from Santa Cruz Biotechnology (sc-32290) and Abcam Inc (ab8049). Rabbit polyclonal anti-IBA (ionized calcium binding adapter) was purchased from Wako (019-19741). Mouse monoclonal anti-GFAP (Glial fibrillary acidic protein) was purchased from EMD Millipore (MAB3402). Anti-mouse IgG, HRP conjugate was purchased from Kang Chen Biotechnology (KC-MM-035). Anti-mouse IgG-FITC and anti-rabbit IgG-Rhodamine were purchased from Jackson Biotechnology (115-095-003, 111-025-003).

As we described previously [28 (link)], the mice were deeply anesthetized with isoflurane, and their ascending aortas were perfused first with PBS and then with 4% paraformaldehyde. After perfusion, the L4–L6 spinal cord segments were collected and then postfixed overnight. The spinal cord sections were sliced at a thickness of 30 µm (free-floating) on a cryostat and processed for immunohistochemistry analysis. The sections were first blocked with 2% goat serum at room temperature for 1 h and then incubated at 4 °C overnight with the following primary antibodies: anti-ATF-3 (rabbit, 1:1000, Santa Cruz Biotechnology Inc.), anti-GFAP (mouse, 1:1000, EMD Millipore), anti-IBA1 (rabbit, 1:1000; Wako Chemicals Inc., USA) and anti-NF200 (mouse, 1:1000, Sigma, catalog: N0142). After washing, the sections were incubated at room temperature for 2 h with the following secondary antibodies (1:400, Jackson ImmunoResearch): Cy3-donkey anti-rabbit (catalog: 711-165-152) and FITC-donkey anti-mouse (catalog: 715-095-150). The stained sections were observed and photographed with a Leica fluorescence microscope.