Gfap antibody

U251 cells were plated in 6-well plates and grown until confluency. The medium was replaced with serum-free ASC-conditioned medium and UC-MSC-conditioned medium and the induction was carried out for two consecutive days. U251 cells were then fixed with 4% paraformaldehyde for 15 min, followed by three washes with phosphate-buffered saline (PBS). After a 1-hour blocking with 0.2% Triton X-100 and 3% goat serum in PBS, the cells were incubated with primary antibodies overnight at 4°C. The primary antibodies used in this study include rabbit polyclonal GFAP antibody (1 : 200 dilution; Santa cruz) and mouse monoclonal Tuj1 (1 : 200 dilution; Millipore); subsequently, the cells were washed with PBS three times and then incubated with Dylight 549 goat anti-rabbit secondary antibody (Jackson Immunoresearch Laboratories) and Dylight 488 goat anti-mouse secondary antibody (Jackson) and finally stained with 4′,6′-diamidino-2-phenylindole hydrochloride (DAPI) for 5 minutes.
For quantitative imaging, fluorescent images of the cells were collected automatically by use of the Cellomics high content screening (HCS) system using a 10x objective lens. For axon outgrowth assays, images of GFAP and DAPI staining were analyzed using Neuronal Profiling BioApplication software (Cellomics).

Immunohistochemistry assay was performed, as in our previous study (Zhang et al., 2017 (link)). Briefly, brain tissue samples were embedded in optimum cutting temperature compound (Sakura Finetek, Torrance, CA, USA) and stored at −80°C. Samples sections were cut into 10-μm slices and antigen retrieval was performed using citrate buffer. Sections were treated with 3% hydrogen peroxide (Sangon Biotech Co., Ltd., Shanghai, China) in PBS for 10 min and then incubated in 5% BSA for 10 min. Sections were incubated overnight at 4°C with primary antibodies as follows: TH (F-11) antibody (1:50; Santa Cruz Biotechnology Inc.), nitrotyrosine (11C2) antibody (1:50; Santa Cruz Biotechnology Inc.), glial fibrillary acidic protein (GFAP) antibody (1:50; Santa Cruz Biotechnology Inc.) and ionized calcium-binding adapter molecule 1 (IBA-1) antibody (1:500; WAKO, Osaka, Japan). After washing 3 times with PBS for 5 min each, sections were incubated sequentially in HRP-conjugated goat anti-mouse and goat anti-rabbit secondary antibody (ZSGB-BIO, Beijing, China) for 2 h at 37°C. Sections were visualized with a 3,3-diaminobenzidine (DAB) peroxidase substrate kit (Boster, Wuhan, China). Integrated option density (IOD) was determined using an Image-Pro Plus 6.0 photogram analysis system (IPP 6.0; Media Cybernetics, Bethesda, MD, USA).

Spinal cords were immediately dissected and placed in 4% PFA for 4 h at 4°C for post-fixation, then transferred in 0.2 M phosphate buffer and left overnight. Next, spinal cords were stored in 30% sucrose for 3 days at 4°C before embedding those into the OCT compound (Tissue-Tek). Embedded blocks were stored at -80°C until cryosectioning using a cryostat (Leica CM 1850). 25 μm transverse sections were made for immunohistochemistry and syrinx confirmation. The monoclonal GFAP antibody (1:200, Santa Cruz Biotechnology, Dallas, TX, USA) was used to identify astrocyte cells around the syrinx. Cell nuclei were stained with 10 mM Hoechst 33342 for 7 min followed by mounting using ProLong™ Gold Antifade (ThermoFisher, Waltham, MA, USA). Images were taken using an Olympus FV 1000 confocal microscope.