Mab3424

After continuous administration of ZTC for 8 days, BrdU was firstly injected twice by 2 h interval; and after 20 days, BrdU was again injected twice by 2 h interval; and after another 24 h, the mice were sacrificed and the brain slices were prepared for immunochemistry experiment. The mouse brain was treated with 4% paraformaldehyde for more than 48 h, and then dehydrated by two steps in 20% and 30% sucrose, separately. Coronal continuous sections, thickness 20 um, each 10 pieces were collected in a small hole in a 24-well plate. Subsequently, different samples were randomly sampled at the same location and stained and the results were counted (Liang et al., 2017 (link)). BrdU staining: Sections were blocked with 1% H₂O₂ in dark at room temperature for half an hour, and melted by 2M HCI at 37°C for 1 h, and the primary antibody (1: 500; Cat. No. MAB3424, Millipore) was incubated at 4°C overnight. GAD67 staining: brain sections were blocked with 0.5% Tween in PBS for 2 h, and diluted with 0.05% Tween in PBS for primary antibody (1: 500; Cat. No. MAB5406, Millipore) and incubated overnight at 4°C. DCX brain sections were blocked with 0.5% Triton-100 in PBS for 2 h, and diluted with 0.3% Triton-100 in PBS for primary antibody (1: 500; Cat. No. 4604, Cell Signaling) and incubated overnight at 4°C.

hDPSCs were incubated with 30 μl BrdU (Sigma, Germany) for 48 h. The solutions were then dispersed, the cells were fixed, DNA was denatured, and anti-BrdU antibody (MAB3424, Millipore, USA) was applied. The secondary antibody used was Alexa fluor 488 goat anti-mouse (A11001, Invitrogen, USA). Fluorescence microscopy was used to view the stained cells.

Embryos were soaked in Tyrode’s solution containing BrdU (final concentration; 0.1 mM, Thermo Fisher, B23151) with or without aphidicolin (SIGMA, A0781) at 37 °C for 15 min, and cultured for 12 hr at 37 °C (BrdU was incorporated for 12 hr). Embryos were then fixed in 4% paraformaldehyde (PFA, Electron Microscopy Sciences)/PBS for 30 min at room temperature (RT). Embryos were then washed with PBS to remove PFA, and unincorporated BrdU, and incubated with 1 M HCl for 1 hr at RT to denature DNA. BrdU signal was detected by immunofluorescence staining with anti-BrdU antibody (1∶200, Millipore, MAB3424).