Alexa fluorr 488 goat anti rat igg secondary antibody h l

1×10⁴ cells were cultured in 24-well plates overnight at 37 °C, 5% CO₂. DMEM medium with different concentration of HHT as added to each well, and cells were cultured at 37 °C for 2 days. Add 10 μg/ml BrdU (Abcam, USA,) and incubation for 2 h. The cells were then washed with PBS and fixed in 4% paraformaldehyde for 30 min. Subsequently, 2M HCl were used to treat the cells 30 min at room temperature. After being washed three times with PBS buffer, the cells were treated with 0.5% Triton X-100 and then blocked with 5% goat serum (ZSGB-Bio, Beijing, China) for 1 h. The cells were incubated with a monoclonal rat primary antibody against BrdU (1:1000) at 4 °C overnight,after being washed three times with PBS buffer and followed by incubation with Alexa FluorR® 488 goat anti-rat IgG secondary antibody (H+L; 1:250, Invitrogen). The nuclei were stained with DAPI (1:500). The percentage of BrdU staining was calculated from at least 10 microscopic fields.

Briefly, 10,000 cells were cultured in 24-well plates overnight at 37 °C. Culture medium containing DMSO or T-96 was added to each well, and cells were cultured at 37 °C °C for 2 days. After incubation with BrdU (Abcam, USA, 10 μg /ml) for 1 h, the cells were washed with phosphate-buffered saline (PBS) and fixed in 3.7% paraformaldehyde for 10 min. Subsequently, the cells were treated with 2 M HCl at room temperature for 10 min and then incubated at 37 °C for 15 min. After being washed three times with PBS buffer, the cells were blocked with 10% goat serum (ZSGB-Bio, Beijing, China) containing 0.5% Triton X-100 at 37 °C for 2 h. Cells were incubated with a monoclonal rat primary antibody against BrdU (1:1000) at 4 °C overnight, followed by incubation with Alexa FluorR® 488 goat anti-rat IgG secondary antibody (H + L; 1:10,000, Invitrogen). The nuclei were stained with DAPI. The percentage of BrdU staining was calculated from at least 10 microscopic fields.