Ba0056

After appropriate survival times, animals were deeply anesthetized with pentobarbital sodium (100 mg/kg, i.p.) and perfused with normal saline, followed by 4% paraformaldehyde (PFA). After the perfusion, the brain was removed and post-fixed in 4% PFA overnight and then dehydrated in 30% sucrose for 48 h. Brain tissue sections (30 μm) were cut in a cryostat (CM 3050S, Leica). After rinsing with 0.5% Triton-X 100 in PBS for 30 min, brain sections were blocked with 5% donkey serum for 2 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: anti-GFAP antibody (rabbit, 1:400, BA0056, Boster), anti-GFAP antibody (mouse, 1:400, G3893, Sigma), anti-NeuN (rabbit, 1:400, MABN140, Millipore), anti-ATP1A2 antibody (rabbit, 1:300, PA5-77512, Invitrogen). After washing, the sections were incubated with Alexa Fluor™ 488/594/647 secondary antibody (1:1000, Molecular Probes) for 2 h at room temperature. After rinsing the sections, we mounted the sections on slides using Vectashield Mounting Media (Vector Labs). The stained sections were examined with a Leica SP8 confocal microscope.

Six weeks after transplantation, the rats were sacrificed by injection of sodium pentobarbital, and the brain tissues were fixed with 4% paraformaldehyde. After being immersed in 15% and 30% sucrose in PBS, the brain tissues were sliced with a freezing microtome (20 μm thick). Cultured cells were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature. The fixed cells and brain sections were then permeabilized with 0.5% Triton X‐100 for 15 min at 37°C, blocked in 10% goat serum for 30 min at 37°C, and incubated overnight at 4°C with primary antibodies. They were then incubated with secondary antibodies in the dark at room temperature for 1 h. Then, the stained samples were counterstained with DAPI (P0131, Beyotime Biotechnology) and observed with a laser scanning confocal microscope. Images were taken with ZEN 2 software. The following antibodies were used: Nestin (1:50, abs137231, Absin, China), NSE (1:50, BA0535, Boster), GFAP (1:50, BA0056, Boster), CCR5 (1:50, sc‐17,833, Santa Cruz Biotechnology), TH (1:500, ab112, Abcam), CCL5 (1:50, sc‐365,826, Santa Cruz Biotechnology), goat anti‐rabbit IgG H&L (Alexa Fluor® 488,1:1000, ab150077, Abcam), goat anti‐mouse IgG H&L (Alexa Fluor® 647,1:1000, ab150119, Abcam), and goat anti‐rabbit IgG H&L (Alexa Fluor® 647,1:1000, ab150079, Abcam).

Well-grown cell spheres were selected for growing on slides coated with polylysine. After drying at 37°C, the slides were washed with phosphate-buffered saline (PBS) three times. The cells were fixed with paraform for 30 min at room temperature, then washed with PBS a further three times. After blocking with 5% goat serum at 37°C for 30 min, primary monoclonal rabbit anti-human nestin (1:30; BA1289, Boster, Wuhan, China) diluted in 1% BSA was added and the cells were placed in a wet box overnight. Subsequently, the cells were washed with PBS, then secondary monoclonal goat anti-rabbit IgG-fluorescein isothiocyanate antibody (1:50; BA1105, Boster) diluted in 1% BSA was added for incubation for 30 min at 37°C. In addition, a negative control in which PBS was used instead of the primary antibody was performed. The slides were observed using an Olympus BX51 fluorescence microscope (Olympus Corporation, Tokyo, Japan). Immunofluorescent assays for glial fibrillary acidic protein (GFAP) and β-tubulin in the differentiated stem cells were conducted using an identical protocol as that used for nestin, with the exception of the respective antibodies. GFAP and β-tubulin were used to identify glioma cells. They were detected by immunofluorescence using monoclonal GAFP (1:30; BA0056; Boster) antibody diluted in 1% BSA and monoclonal β-tubulin (1:30; BM1453; Boster).

After the behavioral experiment, three mice were randomly selected from each group for the immunofluorescence, they were perfused with 0.9% NaCl and 4% paraformaldehyde under isoflurane anesthesia. Mouse brain tissue was fixed in a 4% formaldehyde solution and then dehydrated in a gradient of 20% and 30% sucrose solution. The processed brain tissue is processed into slices with a thickness of 25 microns by a frozen slicer (Leica Microsystems, Wetzlar, Germany). The tissue sections were sequentially reacted with 0.3% TritonX-100, 10% goat serum, target primary and secondary antibodies to complete immunofluorescence staining. The main antibodies are: Anti-ß-amyloid (ab201060, Abcam, 1:200); Anti-GFAP (BA0056, BOSTER, 1:100); Anti-IBA1 (PB0517, BOSTER, 1:100); Goat Anti-Rabbit IgG (BM3894, BOSTER, 1:1000); Dnk pAb to Rb IgE (ab150073, Abcam, 1:1000). Finally the images were imaged using a fluorescent microscope and analysed using ZEN 2.0 software (Olympus, Japan).

After the behavioral experiment, three mice were randomly selected from each group for the immunofluorescence, they were perfused with 0.9% NaCl and 4% paraformaldehyde under isoflurane anesthesia. Mouse brain tissue was fixed in a 4% formaldehyde solution and then dehydrated in a gradient of 20% and 30% sucrose solution. The processed brain tissue is processed into slices with a thickness of 25 microns by a frozen slicer (Leica Microsystems, Wetzlar, Germany). The tissue sections were sequentially reacted with 0.3% TritonX-100, 10% goat serum, target primary and secondary antibodies to complete immunofluorescence staining. The main antibodies are: Anti-ß-amyloid (ab201060, Abcam, 1:200); Anti-GFAP (BA0056, BOSTER, 1:100); Anti-IBA1 (PB0517, BOSTER, 1:100); Goat Anti-Rabbit IgG (BM3894, BOSTER, 1:1000); Dnk pAb to Rb IgE (ab150073, Abcam, 1:1000). Finally the images were imaged using a fluorescent microscope and analysed using ZEN 2.0 software (Olympus, Japan).

GSCs were incubated on polylysine-coated coverslips. After 48 h of treatment with GO, the cells on the coverslips were treated with 4% paraformaldehyde for 30 min. The coverslips were washed with PBS, followed by permeabilization with 0.3% Triton X-100 for 30 min at room temperature. The coverslips were blocked with 10% goat serum. We performed incubation with anti-CD133 (1:200; PB0168, Boster) and anti-GFAP (1:200; BA0056, Boster) antibodies overnight, which was followed by incubation with secondary antibody for 1 h. The nuclear DNA was labeled with DAPI. Representative images were obtained with an IX71 Olympus fluorescence microscope.