Goat anti cxcr4

Neurospheres were attached to a glass slide by using a cytocentrifuge (Cyto-Tek) at 1000 rpm for 3 minutes. Cells were fixed with acetone : methanol 1 : 1 at 4°C, washed with PBS, and treated with 3% BSA for at least 1 hour at room temperature. Cells were then incubated with the following primary antibodies at 4°C overnight: rabbit anti-Ki67 (1 : 1000, Abcam), mouse anti-nestin (1 : 300, Abcam), goat anti-CXCR4 (1 : 300, Abcam), rabbit anti-βIII tubulin (1 : 300, Epitomics), rabbit anti-GFAP (1 : 7000, Abcam), rabbit anti-SOX2 (1 : 300, Abcam), and rabbit anti-CNPase (1 : 1000. Abcam). Secondary antibodies (goat anti-rabbit, goat anti-mouse, and goat anti-rat) were utilized conjugated with Alexa Fluor 594 (1 : 1000, Abcam). Images were taken with confocal laser scanning microscope (LSM780, Carl Zeiss).

As reported previously (Yang et al., 2015 (link)), the rats were anesthetized with chloral hydrate, then perfused with physiological saline, followed by 4% paraformaldehyde in 0.1 M PB solution. The brain was removed and postfixed overnight at 4°C and then immersed in 30% sucrose in 0.1 M PB. Brain tissue were cut into transverse sections (30 μm thick) on CM1900 freezing microtome (Leica, Germany). After blocking with 10% goat serum in PBS, the sections were incubated overnight at 4°C with the following primary antibodies: mouse anti-GFAP (1:500 Millipore), rabbit anti-Iba-1 (1:500, WAKO), mouse anti-NeuN (1:200, Millipore), Goat anti-CXCR4 (1:200, Abcam) and rabbit anti-c-Fos (1:500, Abcam). On the following day, Cy3- or FITC-conjugated secondary antibodies were incubated for 2–3 h at room temperature. For double immunostaining, sections were incubated with a mixture of primary antibodies overnight at 4°C, followed by a mixture of secondary antibodies. The images were examined under a laser scan confocal fluorescent microscope (Olympus FV1000, Japan).