Alexa fluor 594 secondary antibody h l

Cell proliferation was monitored by BrdU staining. Firstly, 2 × 10⁴ cells were cultured in 24-well plates. 48 h later, the cells were incubated with 10 μg/ml BrdU for 35 min and subsequently fixed with 4% paraformaldehyde (PFA) for 20 min. Then, the cells were treated with 2 M HCl for 10 min, permeabilized with 0.5% Triton X-100 for 10 min, blocked with 10% goat serum for 1 h, and incubated a monoclonal rat primary antibody against BrdU (1 : 300, Sigma-Aldrich) overnight at 4°C. Alexa Fluor® 594 secondary antibody (H+L; Invitrogen) was incubated with the cells at room temperature for 2 h, followed by nuclear staining with DAPI (300 nM). Finally, BrdU-positive cells in random fields were counted under the microscopy.

In total, 2 × 10⁴ cells were cultured in 24-well plates for BrdU staining experiments. The cells were incubated for 35 min with 10 µg/ml BrdU. The cells were fixed with 4% paraformaldehyde (PFA) for 20 min. Cells were treated with 1 mol/L HCL and blocked with 5% goat serum and 0.3% BSA for 2 h. Then, the cells were incubated with a primary antibody against BrdU (Abcam, Cambridge, MA, USA) overnight at 4 °C. Then, an Alexa Fluor^® 594 secondary antibody (H + L; Invitrogen) was incubated with the cells at room temperature for 2 h, and nuclear staining was then performed by incubating with DAPI (300 nM). Finally, the BrdU incorporation rate was calculated from at least ten randomly chosen microscopic fields.

For BrdU staining, 1 × 10⁴ cells were cultured on coverslips in 24-well plates. The cells were incubated with 10 μg/mL BrdU (Sigma) for 30 min, washed with phosphate-buffered saline (PBS), and fixed with 4% paraformaldehyde for 15 min. Then, samples were blocked with 5% goat serum for 2 h, followed by incubation with a primary antibody against BrdU (1:200, ab6326, Abcam, Cambridge, MA, USA) for 1 h, and then an Alexa Fluor^® 594 secondary antibody (H + L; Invitrogen). DAPI (4′,6-diamidino-2-phenylindole) (300 nM) was used for nuclear staining; the percentage of BrdU incorporation was calculated from at least 10 randomly selected fields.