Alexa fluor 488 anti mouse or 594 anti rabbit

For immunofluorescence, cells or tissue sections were incubated with 0.2% Triton X-100 (Gibco) for 30 min and then were washed with PBS and incubated with blocking buffer composed of 3% BSA (Sigma-Aldrich, A9647) in PBS for 30 min at room temperature. The samples were then incubated with primary antibody overnight at 4 °C (SOX2 (Millipore, MAB5603, 1:400), PAX6 (RD, MAB1260, 1:1000); SOX1 (R&D, AF3369, 1: 400), NESTIN (Millipore, MAB5922, 1:400); MAP-2 (Millipore, MAB5622,1:600); GFAP (Sigma, G9269, 1:2000), DCX (Millipore, MABN707, 1:500), TUJ1 (Covance, MRB435P, 1:1000), NeuN (Millipore, ABN78,1:500), P-CREB (CST, 9198)). The following day, the samples were washed with PBS and incubated with the corresponding secondary antibody (Alexa Fluor 488 anti-mouse or 594 anti-rabbit (Invitrogen,1: 500)) for one hour at room temperature. Nuclei were counterstained with DAPI (Sigma-Aldrich, 32,670) for 15 min at room temperature. The cells were photographed using a confocal microscope (Carl Zeiss LSM710 Confocal). Tissue sections were photographed using a microscope (Leica, SP8) and the number of cells was counted using Image J software. The percentage of positive cells was calculated from the total nucleus population. All studies were performed for a minimum of 3 sections per sample, with 6 animals in each group.

For immunofluorescence, the brains were cut into 20 μm slices using a low-temperature thermostat. After incubation with 0.2% Triton X-100 (Thermo Fisher Scientific, 85111, Waltham, MA, USA) for 30 min, the sections were washed with PBS and incubated with blocking buffer composed of 3% BSA (Sigma-Aldrich, A9647) in PBS for 30 min at room temperature. The samples were then incubated with primary antibodies overnight at 4 °C. On the next day, the samples were rinsed with PBS and incubated for one hour at room temperature with the corresponding secondary antibody (Alexa Fluor 488 anti-mouse or 594 anti-rabbit (1:500) (Invitrogen, A30629 and A30678, Waltham, MA, USA). The sections were blocked by anti-fluorescence quenching blocking solution with DAPI, then analyzed and photographed using a microscope (Carl Zeiss Confocal, LSM710, Oberkochen, Germany). The number of cells was counted using Image J software. The percentage of positive cells was calculated from the total nucleus population. All studies were performed three times, with 10 animals in each group. The information of all antibodies is shown in Table 1.