Goat anti rabbit igg pe

On day 23, mice of each group were intraperitoneally administrated with bromodeoxyuridine (BrdU) (75 mg/kg; Abcam, Cambridge, UK) every 2 hours for 12 hours. Two hours after the last injection of BrdU, the mice were anesthetized with diethyl ether. Blood in the brains was flushed out using normal saline through a heart perfusion, and the brains were fixed using 4% paraformaldehyde (Guangzhou Chemical Reagent Factory, Guangzhou, China), also using heart perfusion. Subsequently, the fixed brains were extracted and continued to fix in a fresh 4% paraformaldehyde for 1 week. The fixed brains were paraffin-embedded and sectioned.
After the denaturation of DNA and antigen retrieval, sections were incubated with a mixture of primary antibody BrdU (1:1400; a marker of newly generated cells) and doublecortin (DCX) (1:400; a marker of immature neurons) (Abcam, Cambridge, UK) at 4°C overnight. The next day, sections were incubated with goat anti-mouse IgG-Alexa 488 (1:600) and goat anti-rabbit IgG-PE (1:400) (Invitrogen, CA, USA) in the dark for 2 hours. DNA in the nucleus was stained by DAPI dye (1:1298) (Boyotime Institute of Biotechnology, Haimen, China) for 5 minutes. Finally, sections were mounted using anti-fade mounting medium, and then stored at 4°C in the dark. A section incubated without primary antibodies was considered as a negative control (Additional Figure 1).

For flow cytometry studies of protein marker expression, cells were dissociated into single cells by incubating them with 0.25% trypsin (Gibco) for 5 min at 37 °C and washed with 1x PBS twice. The dissociated cells were fixed with 75% ethanol at 4 °C overnight. Then, the cells were permeabilized with 0.05% Triton X-100 for 3 min and rinsed with 1x PBS twice. The cell number was adjusted to 1 × 10⁶ cells per tube in 700 μL of 1x PBS for each protein marker. The cells were incubated with primary antibodies including: OCT-4 (1:500 dilution, Cell Signaling Technologies), SSEA-4 (1:500 dilution, Cell Signaling Technologies), TUJ-1 (1:500; R&D Systems, Minneapolis, MN, USA), or cTnT (1:500 dilution, Cell Signaling Technologies) at 4 °C overnight. Afterwards, the samples were rinsed with 1x PBS thrice before incubation with secondary antibodies (goat anti-mouse IgG FITC and goat anti-rabbit IgG PE, Invitrogen) at 1:1000 dilution for 1 h at RT. The samples were then washed with 1x PBS twice before analysis using a BD FACSCelesta (Franklin Lakes, NJ, USA) flow cytometer. The cells incubated with secondary antibody but no primary antibodies were processed and washed in the same way for analysis to serve as the negative/isotype control. The resultant data was analyzed with the BD Flowjo software (v10).