Brdu antibody

The cells were assigned to the following groups: control group, ketamine group, and 17β-estradiol group. No drug treatment was added to the control group. NSCs in the ketamine group were exposed to 100 μM ketamine for 24 h. NSCs in the 17β-estradiol group were pretreated with 17β-estradiol (100 nM) for 30 min and then 100 μM of ketamine was added to the culture medium for 24 h. For proliferative analysis, NSCs were seeded on cover slips which were pre-coated with 100 μg/mL poly-L-lysine and incubated with BrdU for the last 4 h. Following being fixed with 4% paraformaldehyde, the cells were stained with BrdU antibody (1:200, Abcam, United Kingdom) and DAPI. As for neuronal differentiation analysis, after being exposed to ketamine with or without 17β-estradiol for 24 h, the cells were seeded on cover slips which were pre-coated with 100 μg/mL poly-L-lysine and incubated with differentiating medium for 7 days, then the cells were harvested for immunohistochemical staining. The cells were labeled with β-tubulin III antibody (1:500; Sigma-Aldrich Inc. St. Louis, MO, United States). Briefly, 5–7 randomly selected fields were captured in each coverslip, and the numbers of β-tubulin III-positive cells were counted (at least 200 cells per test case). Data were collected from three independent experiments.

Six kinds of HNSCC cells (CAL33, CNE-2, S18, S26, AMC-HN-8, TU686) were separately seeded on slides and cultured at 37 °C and 5% CO₂. After drug treatment, EdU (final concentration: 10 μM; Sigma, St. Louis, MO, USA) was added to the medium and incubated for 1 h. The cells were fixed with 4% paraformaldehyde, treated with 2N HCl at 37 °C for 30 min, and sealed and made permeable with blocking solution (BS) (5% goat serum, 1% bovine serum albumin, 0.4% Triton X-100). BrdU antibody (Abcam, Cambridge, UK, 1:200 dilution) was incubated overnight at 4 °C, followed by secondary antibody Alexa Fluor 488 (Invitrogen, Carlsbad, CA, USA, 1:200) for 1 h at room temperature and DAPI for 20 min. A mounting medium (Sigma, St. Louis, MO, USA) was used to seal the plates. Finally, the images were observed under a fluorescence microscope (ZEISS, Oberkochen, Germany).

The primary isolated hepatocytes were seeded at the 6-well plate with supplementary of different concentrations of cytokines including TGF-β, VEGF, HGF and IL-10. These hepatocytes were maintained culturement with the respective cytokines for set time points (day 1, day 3, day 7, day 11 and day 15), and the BrdU (50 µM, Abcam) was added for 12 h befored the fixation. Then, these cells were fixed and immunostained with the BrdU antibody (1:2000, Abcam) according to the manufacturer’s instruction. Subsequently, the PE-coupled Rabbit anti-mouse secondary antibody (1:150, Abcam) was used for staining the BrdU-positive cells and the DAPI was used for nuclei counterstain. The proliferation ablility of hepatocytes were observed and photographed under the fluorescence microscopye (Olympus). Finally, the percentage of BrdU positive cells was evaluated at set time points.

2 × 10⁴ gastric cancer cells were seeded in 24-well plates and then incubated at 37°C in an incubator overnight. After incubating with 10 μg/ml BrdU (Sigma-Aldrich, USA) at 37°C for 2 h, cells were fixed with 4% paraformaldehyde for 15 min. Cells were then treated with 2 M HCL and then with 0.3% TritonX-100 before blocked with 10% goat serum. Successively, cells were exposed to the BrdU antibody (Abcam, Cambridge, USA) and then with the secondary antibody (1 : 2000). Before captured by a microscope, cells were counterstained with Hoechst (1 : 2000).

Cells were seeded in 96-well plates at an initial density of 2× 103 cells/well with 10 μmol/L BrdU solution. After incubation for 2 h, PBS with 4% paraformaldehyde was used to fix the cells for 15 min. Washed cells with PBS and treated with DNase (Tiangen, China) for another 15 min at room temperature. Cells were washed with PBS again and stained with BrdU antibody (Abcam, Cambridge, Massachusetts, USA) for about 8 h at 4°C. Incubated the cells with secondary antibody at room temperature for 1 h. Then dyed cell nucleus with DAPI. Counted the BrdU-positive labeled cells in each group for at least 5 fields.