Goat anti rat cy3

Following the treatment, NSPCs or brain tissues were fixed using 4% paraformaldehyde at room temperature for 1h. After being blocked in 1% BSA, 5% goat serum and 0.2% Triton X-100 in PBS (PH 7.4) at 4°C for 2h, the specimens were incubated with with corresponding antibodies against against Ki67(1:1000, Abcam, UK), DCX (1:500, Abcam, UK), BrdU (1:500, Millipore, Germany), GFAP(1:500, Abcam, UK), Nestin (1:1000, Proteintech Group, Inc, US) at 4°C overnight. Then, cells were incubated with corresponding secondary antibody, goat anti-rabbit Alexa Fluor 488, goat anti-rabbit Alexa Fluor 555, goat anti-rabbit Alexa Fluor 647, Goat Anti-Rat Cy3 or anti-mouse Alexa Fluor 488 (1:200, Beyotime, China) at room temperature for 2h, followed by counterstaining with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI, 2 mg/ml in PBS) for 10 min. Finally, the images were captured using a Zeiss UV 780 Meta confocal microscope (Carl Zeiss) and examined by Adobe Photoshop CS6 software.
For BrdU immunostaining, brain sections were incubated in 2N HCl at 37°C for 30min. Stained sections were rinsed using 0.1M borate solution (pH=8.5) twice for 10min, incubated in 3% H₂O₂ for 30 min and blocked with 5% normal goat serum at room temperature for 1h.

The tissues were fixed in 4% (w/v) paraformaldehyde, dehydrated by treating with an ethanol gradient, embedded in paraffin and cut into sections with a thickness of 7 μm. Immunostaining was performed as follows. The paraffinized tissue sections were rehydrated, and the antigens were retrieved, followed by rinsing three times with PBS. Next, the sections were treated with blocking solution (1% BSA) for 30 min before overnight treatment with primary antibodies at 4℃. Rabbit anti‐mouse antibodies against KI67 (Abcam, ab16667) and CD45 (BD Biosciences, 555 483) and rat anti‐mouse antibodies against CD19 (Biolegend, 115 525) and F4/80 (Biolegend, 123 121) were used as primary antibodies to detect cell proliferation and immune cell infiltration. The secondary antibodies goat anti‐rabbit Alexa Fluor 488 (Invitrogen, A11008), goat anti‐rabbit Alexa Fluor 546 (Invitrogen, A21430‐f) and goat anti‐rat CY3 (Beyotime Institute of Biotechnology, A0507) and DAPI (Beyotime Institute of Biotechnology, China) were used to visualize the respective primary antibodies and cell nuclei. All procedures were performed according to the manufacturer's instructions.