Mouse anti brdu monoclonal antibody

BrdU assay was performed in 6-well plates at 25 °C. BrdU (BBI Life Sciences, China; Cat. No. E607203) was dissolved in DMSO to prepare a 25-mM storage solution and stored at − 20 °C. Before use, the BrdU storage solution was diluted into a 1 mM solution using DMSO. At 6 or 7 hpf, BrdU was added to FSW to a final concentration of 1 μM. In control groups, the same volumes of DMSO was added. After one or two hours, the samples were collected at 8 hpf, and the embryos of BrdU and control groups showed no detectable morphological changes at the end of the treatment. The embryos were washed with FSW, fixed in 4% PFA at room temperature for 30–60 min, transferred to PBST and stored at 4 °C. Before immunostaining, the samples were incubated in 1 M HCl for 30 min to denature DNA. After washing with PBST, the samples were incubated in the blocking buffer (2% BSA in PBST) for 2 h at RT. The primary (anti-BrdU mouse monoclonal antibody [Sigma-Aldrich], 1:200) and second (Alexa Fluor 488 goat anti-mouse antibody [PTC], 1:200) antibodies were applied to detect the incorporated BrdU in the nuclei.

The crayfish were injected with 10 μL/g fresh weight of 50 mM BrdU dissolved in crayfish saline (CFS:0.2 M NaCl, 5.4 mM KCl, 10 mM CaCl2.2H2O, 2.6 mM MgCl2.6H2O, 2 mM NaHCO3, pH 6.8). At 3 h post injection the stomach covered with the HPT was dissected from crayfish and immediately fixed in 4% paraformaldehyde in PBS and kept at 4°C overnight. Then the HPT was carefully removed from the stomach, dehydrated through an ethanol series 50%, 70%, 90%, 100%, and xylene, and finally embedded into paraffin blocks. The HPT was then sectioned vertical sections from anterior to posterior with thickness of 10 mM and the sections were placed on MENZEL Super frost glass slides for further processing. After deparaffination and rehydration the slides were washed with CPBS-TB (CPBS:10 mM Na₂HPO₄, 10 mM KH₂PO₄, 0.15 M NaCl, 10 μM CaCl₂, 10 μM MnCl_2, 2.7 μM KCl; pH 6.8, containing 0.5% Tween 20 and 0.5% BSA). The slides were then incubated in 2 N HCl containing 0.mg/mL pepsin for 30 min at 30°C. After washing 5 times with CPBS-TB, the slides were incubated in anti-BrdU mouse monoclonal antibody (Sigma) dissolved in CPBS-TB for 30 min at RT, followed by secondary antibody FITC-labeled anti-mouse IgG (Sigma) in CPBS-TB for 30 min at RT. Finally, the nuclei were stained with propidium iodide (10 μg/mL) and mounted in Vectashield.

For cell cycle progression analysis cells that were pulsed with BrdU and fixed with 70% ethanol were washed twice with PBS at room temperature. Cells were incubated with 1 mL 2 M HCl (containing 0.1 mg/ml pepsin) for 20 min at room temperature. Cells were washed three times with PBS-T (PBS, 0.2% tween20, 1% goat serum). After the third PBS-T wash, cells were resuspended in 100 μL PBS-T and incubated with 2 μl mouse monoclonal anti-BrdU antibody (Sigma-Aldrich) for 1 h at room temperature. Cells were washed twice with PBS-T, resuspended in 100 μL PBS-T and incubated with 2.5 μL of FITC-conjugated rabbit anti-mouse immunoglobulins (Sigma-Aldrich) in the dark for 1 h at room temperature. Cells were then washed once with PBS-T and resuspended in 0.5 mL PI/RNase A solution (RNaseA 0.1 mg/ml, Propidium iodide 25 μg/ml). Cells were incubated for 5-15 min at 37°C or kept at 4°C overnight before analysis on an Accuri C6 flow cytometer. FACS profiles were analyzed by BD Accuri C6 software.