Anti brdu antibody

For BrdU incorporation assay, cells were labeled with 10 μm BrdU (Roche, Indianapolis, IN, USA) for 2 h, fixed with 4% para-formaldehyde, and immunostained with anti-BrdU antibody (Roche) followed by staining with Cy™3-conjugated goat anti-mouse IgG (115-165-146; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and counter-stained with DAPI. BrdU-positive cells were scored under a fluorescent microscope and presented as the percentage of BrdU-positive nuclei over total number of nuclei counted. At least 300 nuclei were counted. For immunofluorescence, cells were fixed with 4% paraformaldehyde, immunostained with primary and secondary antibodies in 4% BSA, and counter-stained with DAPI. Antibodies used include anti-p53 (DO-1; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and goat anti-mouse Alexa Fluor 488 (A11001; Santa Cruz Biotechnology). Cell images were recorded with an Axiovert 200M microscope (Carl Zeiss, Oberkochen, Germany) and analyzed with axiovision 3.1 software (Carl Zeiss).

The cells were spiked with BrdU (Roche) at 1:200 for 2 h in TC-100 insect medium. Then, the cells were fixed in 4% paraformaldehyde for 15 min and washed three times with phosphate-buffered saline containing 5% Tween-20 (PBST, Beyotime, Shanghai, China). Then, the cells were blocked with 3% bovine serum albumin and 10% sheep serum in PBS (blocking solution) at 37 °C for 1 h. The cells were further incubated with anti-BrdU antibody (1:200; Roche) and anti-HA antibody (1:200; Abcam, Cambridgeshire, UK) in blocking solution for 1.5 h at 37 °C. Then, they were washed six times with PBST for 6 min each time and then incubated for 1 h with Alexa Fluor 555-conjugated donkey anti-rabbit IgG secondary antibody (1:500; Life Technologies, Rockville, MD, USA) and Alexa Fluor 488-conjugated donkey anti-mouse IgG secondary antibody (1:500; Life Technologies) in blocking solution. The cells were observed under a confocal microscope (Olympus, Tokyo, Japan).

5 ×10⁷ DT40 cells were pulse-labeled with 20 μM BrdU for 10 min. Cells were then harvested and washed and resuspended in medium either with CPT (20 μM) or without CPT and further cultured for 15 min. Partial digestion of chromatin DNA with Micrococcal nuclease (MNase) was performed as described previously [31 (link)–33 (link)], with slight modifications. Briefly, the above-mentioned BrdU-labeled cells were suspended in 0.5 ml of lysis buffer (18% Ficoll 400, 10 mM KH₂PO₄, 10 mM K₂HPO₄, 1 mM MgCl₂, 0.25 mM EGTA, 0.25 mM EDTA, and 1 mM Pefabloc SC [Roche, Mannheim, Germany]). After centrifugation at 14,000 rpm for 30 min at 4°C, the crude chromatin fraction was resuspended in 0.3 ml of buffer A (10 mM Tris-HCl [pH 8.0], 150 mM NaCl, 5 mM KCl, and 1 mM EDTA) containing a proteinase inhibitor cocktail (Complete, Roche). After addition of CaCl₂ (5 mM final concentration), 0.1 ml aliquots of crude chromatin suspension were digested with several different amounts of MNase (0, 5, 10, and 20 U/ml) at 37°C for 5 min. The reaction was terminated by adding 25 mM EDTA, and DNA was purified. DNA samples were resolved in 2% agarose gel electrophoresis followed by membrane transfer and immunodetection using anti-BrdU antibody (Roche).

Live zebrafish embryos injected with BrdU were fixed in 4% paraformaldehyde (PFA) overnight and then replaced with methanol at −20 °C for 2 hpf. Embryos were permeabilized with proteinase K, refixed in 4% PFA for 20 min and then rinsed in PBST, followed by incubation in 2 n HCl for 1 h. Then, embryos were blocked for 1 h in blocking solution and incubated with primary anti-BrdU antibody (Roche, Mannheim, Germany) and secondary Alexa Fluor 488-conjugated anti-mouse antibody (Invitrogen). The BrdU-labeled embryos were treated with 30% sucrose and then washed three times using PBST and embedded in O.C.T. medium (SAKURA, Torrance, CA, USA). The embryos were sectioned using LEICACM1900 cryostats. Detection of both cmyb RNA and mitosis marker pH3 and BrdU simultaneously was conducted as described previously [59 (link)].
Embryos at 24 hpf in methanol were washed with PBST and then permeabilized with proteinase K. After being washed with PBST, embryos were subjected to TUNEL assay according to the instruction of the TUNEL staining kit (Roche) at 4 °C overnight.

5 dpf larvae were incubated in 15% DMSO and 85% hanks (Gibco) solution with 10 mM bromodeoxyuridine (BrdU, Sigma) for 20min on ice. Then larvae were fixed in 4% paraformaldehyde (PFA) at 4°C overnight. After gradual dehydration and rehydration in methanol, larvae were permeabilized 10 min with 100% acetone prechilled to −20°C, and then 20 min with 0.1% trisodium citrate(with 0.1% Triton X-100) at RT. Larvae were rinsed three times with ddH₂O and incubated in 2N HCl for 1h at RT. Subsequently larvae were washed in PBST (0.1% Triton X-100 in PBS) several times and blocked in 2% goat serum for 2h at RT. Then whole-mount immunofluorescence was performed with 1:10 anti-BrdU antibody (Roche) at 4°C overnight followed by incubation with secondary antibodies conjugated to fluorescein (1:1000) according to standard protocols. BrdU labeled larvae were embedded in 1.0% low-melting point agarose for imaging. Images were performed on an Olympus FV1000-MPE laser scanning confocal microscope. The number of BrdU positive nuclei was counted over 11 somites of the fish trunk between the anus and head.

To characterize DNA replication, 10 mM bromodeoxyuridine (BrdU) (Roche, Basel, Switzerland) was added to the growth medium for 15 min. Subsequently, the cells were washed three times with PBS, fixed in 3% paraformaldehyde (Sigma) for 20 minutes at −20 °C, washed three times with PBS, and stained using anti-BrdU antibody (Roche) primary antibody, FITC–conjugated secondary antibody (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA), and Hoechst nuclear stain (Sigma).