For detection of pregranulosa cell proliferation, bromodeoxyuridine (BrdU, B5002; Sigma) was added to the medium to 10 μM and incubated for 1 h at 37°C. The ovaries were fixed in 4% paraformaldehyde overnight at 4°C for immunofluorescence analysis.
Brdu b5002
Manufactured by Merck Group
Sourced in United States
BrdU (B5002) is a nucleoside analog of thymidine that can be incorporated into the DNA of dividing cells as a substitute for thymidine. It is commonly used as a cell proliferation marker in biological research.
Automatically generated - may contain errors
Lab products found in correlation
Piceatannol, by Merck Group (2 mentions)
β catenin 610153, by BD (1 mentions)
Notch 1 ab52627, by Abcam (1 mentions)
Bromodeoxyuridine (brdu), by Merck Group (1 mentions)
6 well culture dishes, by NEST Biotechnology (1 mentions)
Anti brdu fitc antibody, by BD (1 mentions)
Facscalibur flow cytometer, by Beckman Coulter (1 mentions)
P3972, by Merck Group (1 mentions)
Hm 325 rotary microtome, by Thermo Fisher Scientific (1 mentions)
Cm1950 cryostat, by Leica (1 mentions)
16 protocols using brdu b5002
1
Detecting Pregranulosa Cell Proliferation
2
Immunohistochemical Profiling of Cellular Markers
3
Bromodeoxyuridine (BrdU) Injection Protocol
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Bromodeoxyuridine (BrdU-B5002, Sigma-Aldrich) was intraperitoneally (i.p.) injected (50 mg/Kg of animal weight) immediately after the preparation of a solution of 50 mg/mL, 10% in dimethyl sulfoxide (DMSO) [8] (link). Injections were performed twice daily, beginning at the day of CFA or CFA vehicle intra-articular injection (day 0), until 24 h prior to animals sacrifice (to allow BrdU clearance) by intracardiac perfusion, as described below. The following experimental groups were used: controls (CFA-vehicle non-inflamed rats injected with BrdU until day 3 and sacrificed at day 4; N = 6 rats), 4d MA (CFA-inflamed rats injected with BrdU until day 3 and sacrificed with 4d of disease; N = 5 rats) and 7d of MA (CFA-inflamed rats injected with BrdU until day 6 and sacrificed with 7d of disease; N = 6 rats). Prior to these experiments, a group of naive animals was injected twice a day, with 10% DMSO solution i.p., for 6 days and no toxic effects or signs of peritoneal inflammation were found (data not shown).
4
Primordial Folliculogenesis Regulation
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Ovaries were separated from mouse ovarian capsules in prechilled PBS (10 mM, pH 7.4). The isolated ovaries were cultured in 6-well culture dishes (NEST Biotechnology) with 1.1 ml of basic DMEM/F12 (Gibco, Life Technologies) at 37°C in a 5% CO2/95% air atmosphere with saturated humidity. Half of the medium was replaced every two days until the ovaries grew to the required stage. To determine the role of SP1 in primordial folliculogenesis, ovaries were treated with lentivirus containing shRNA (at 15.5 dpc) or with the SP1 inhibitor MIT (100 nM; M6891; Sigma-Aldrich) (at 16.5 dpc). For the BrdU incorporation assay, ovaries were treated with BrdU (B5002; Sigma-Aldrich) for 1 h before being collected for analysis.
5
BrdU Labeling of Proliferating Cells
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We conducted this experiment using a method described recently [43 (link), 44 (link)]. Briefly, BrdU (B5002, Sigma-Aldrich) was intraperitoneally injected to dams at E17.5 or pups at P0 with the concentration of 100 mg/kg. Brains were collected 30 min after the injection for E17.5 or 2 h after the injection for P0.
6
Cell Cycle Analysis by Flow Cytometry
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For cell cycle analysis, two-color flow-cytometric analysis was used for accurate determination of the cell cycle profile. Mock-infected and infected cells were pulsed with bromodeoxyuridine (BrdU [B5002; Sigma, Saint Louis, MO, USA] 10 μM to approximately 1 × 106 cells) for 1 h prior to harvesting with trypsin. Cells were fixed with ice-cold 70% ethanol at 4 ℃ overnight and then treated with 2 N HCl containing 0.5% Triton X-100 for 30 min. Residual acid was neutralized by incubating the cell suspension with 0.1 M sodium borate (pH 8.5) for 2 min at room temperature. Cells were then incubated with anti-BrdU-FITC solution (anti-BrdU-FITC antibody [556028; BD Biosciences Pharmingen, San Diego, CA, USA] in a 1:5 dilution) at 4 ℃ overnight. The cell suspension was incubated with propidium iodide (PI) staining solution in phosphate buffered saline (PBS) (50 μg/ml PI [Sigma, Saint Louis, MO, USA] and 200 μg/ml RNase [Beyotime, Shanghai, China]) for 30 min at 37 ℃ and then analyzed with a FACSCalibur Flow Cytometer (Beckman, Mississauga, ON, Canada) and FlowJo software.
7
BrdU Proliferation Assay in Mice
8
Tbr2-CreER(T2) Mice Embryo Labeling
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For Tbr2‐CreERT2 mice, pregnant dams were intraperitoneally administered tamoxifen (TM; T5648, Sigma‐Aldrich; 10 mg/kg) and progesterone (P3972, Sigma‐Aldrich; 5 mg/kg) dissolved in corn oil. Embryos were acquired at E18.5 through caesarean section, and they were fostered by other synchronous lactating females. Brains were collected at P7 for further analysis. Fresh bromodeoxyuridine (BrdU, B5002, Sigma‐Aldrich) or idoxuridine (IdU, 1336001, USP) solution (concentration 50 mg/kg) was injected intraperitoneally into pregnant females at the indicated embryonic ages.
9
Nanoparticle-Mediated Gene Silencing in Zebrafish
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siRNAs were encapsulated in polyethylene glycol–polylactic acid nanoparticles using a double emulsion-solvent evaporation technique and then injected into the pericardial sac40 (link)65 66 (link). Briefly, zebrafish were allowed to recover for 1 day after ventricular resection. To evaluate the effect of siRNA on its target gene expression, the hearts were collected at 2 d.p.a., and total RNA was isolated to assess the expression of the respective genes by quantitative RT–PCR. To evaluate the effect of genes on cardiomyocyte proliferation, 50 μl polyethylene glycol–polylactic acid nanoparticle-encapsulated siRNAs was injected first, and ∼1 h later, 50 μl 2.5 mg ml−1 BrdU (B5002; Sigma) was injected into the thoracic cavity daily from 7 to 14 d.p.a. The hearts at 14 d.p.a. were collected for subsequent experiments. siRNA sequences for cdkn1a, cdkn1c and dnmt3ab are shown in Supplementary Table 2 .
10
Isolation and Culture of Neonatal Rat Cardiomyocytes and Fibroblasts
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Cardiomyocytes (CMs) and cardiac fibroblasts (CFs) were harvested from 1‐ to 3‐day‐old neonatal rats. Briefly, rat's heart was quickly removed and 0.04% collagenase II was employed to digest the minced ventricles for six cycles. Cells were harvested and suspended in DMEM medium enriched with 10% FBS. CMs and CFs were collected by differential centrifugation. After 48 hours of culture, BrdU (b5002, sigma Aldrich, United States) was added to the CMs to remove a small amount of residual CFs. CFs were cultured continuously and passaged normally. The second generation was used for experiment.
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