Animals were injected with 5 shots of 32 nL each of BrdU dissolved in 10% DMSO at a concentration of 10 mg/mL using a Nanojet microinjector (Drummond). Five independent samples (each of 3 animals) for experimental class were treated in 2% HCl in 5/8 Holtfreter for 5 min at 4°C, 24 hours after BrdU injection. Animals were then dissociated in single cells by using 1:1:13 parts of glycerol, acetic acid and dH2O for 24 hours at 4°C. BrdU detection was performed as previously described [24 (link)] using anti-BrdU antibody (Anti-BrdU B44, BD, 1:50 dilution) in 10% FBS for 1 hour. After washes, slides were incubated with 1:200 goat anti-mouse Alexa Fluor 488 in 10% FBS for 30 min and then mounted in 80% glycerol containing the nuclear dye Hoechst 33342 (Molecular Probes). BrdU- and Hoechst 33342-positive nuclei were counted using a Axioplan microscope (Carl Zeiss Microscopy GmbH). Three independent experiments were performed.
Anti brdu b44
Manufactured by BD
Sourced in United States
Anti-BrdU B44 is a monoclonal antibody that specifically binds to bromodeoxyuridine (BrdU), a synthetic nucleoside that is incorporated into the DNA of dividing cells. This antibody can be used in various immunological techniques, such as immunohistochemistry and flow cytometry, to detect and quantify cells that have incorporated BrdU during DNA synthesis.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Axioplan microscope, by Zeiss (1 mentions)
Hoechst 33342, by Zeiss (1 mentions)
Rnase a, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Facsverse, by BD (1 mentions)
5 bromo 2 deoxyuridine, by Merck Group (1 mentions)
Alexa fluor 488 anti mouse igg1, by Thermo Fisher Scientific (1 mentions)
Mab3034, by Merck Group (1 mentions)
Sp5 confocal microscope, by Leica (1 mentions)
90i fluorescence microscope, by Nikon (1 mentions)
Alexa fluor 594 anti mouse, by Thermo Fisher Scientific (1 mentions)
Anti laminb1 clone b10, by Santa Cruz Biotechnology (1 mentions)
Anti rad51 clone epr4030 3, by Abcam (1 mentions)
Alexa fluor 594, by Proteintech (1 mentions)
Vectashield containing dapi, by Vector Laboratories (1 mentions)
6 protocols using anti brdu b44
1
BrdU Labeling and Detection in Cells
2
Immunofluorescence of BrdU and RPA
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Immunofluorescence was performed on cells grown on coverslips. For BrdU immunofluorescence, BrdU-pulse labeled cells were first fixed, permeabilized and then treated with DNaseI (100 U/ml, New England Bioscience/NEB, Ipswich, MA, USA) at 37 °C for 30 min prior to incubation with Anti-BrdU (B44, #347580, BD Biosciences, San Jose, CA, USA). For RPA staining, before fixation, cells were subjected to in situ cell fractionation as previously described.44 (link)
3
Cell Cycle Analysis by Flow Cytometry
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Cells were fixed with 70% ethanol and stained with 10 µg/ml propidium iodide (Sigma-Aldrich, Saint-Louis, MO, USA) containing 100 µg/mL RNase A (Invitrogen, Carlsbad, CA, USA). For Bromodeoxyuridine (BrdU) analysis, cells were incubated with 75 µM 5’-Bromo-2’-Deoxyuridine (Sigma-Aldrich, Saint-Louis, MO, USA) for 2 h and further processed via BD Biosciences protocol (Anti-BrdU B44). Cell cycle distribution was assessed with BD FACSVerse (Piscataway, NJ, USA).
4
DNA Fiber Assay for Replication Dynamics
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Exponentially growing cells were pulsed with 30 μM CldU (Merk-Milipore) for 30 min and then with 250 μM IdU (Merk-Milipore) for another 30 min. Cells were then lysed on the slide by adding spreading buffer (0.5% SDS, 200 mM Tris pH 7.4, 50 mM EDTA) and incubated for 6 min at RT in humidity chamber. DNA fibers were stretched by tilting the slide ∼30° and, after air drying, fixed for with ice-cold 3:1 methanol:acetic acid solution. Slides were then incubated in 2.5 HCl for 30 min at RT, and blocked with PBS + 1% BSA + 0.1% Triton X-100 before incubation with anti-CldU (anti-BrdU, sc-56258 BU 1/75 ICR1 from Santa Cruz Biotechnology, RRID: AB_305426) and anti-IdU (anti-BrdU, B44 from BD Bioscience, RRID:AB_2313824 ) antibodies overnight at 4°C. The slides were then incubated with anti-rat IgG Alexa Fluor 555 (Thermo Fisher Scientific) and anti-mouse IgG1 Alexa Fluor 488 (Thermo Fisher Scientific). Finally, they were incubated with anti-ssDNA (MAB3034 from Merck Millipore) and anti-mouse IgG2a Alexa Fluor 647 (Thermo Fisher Scientific) and mounted with Prolong (Thermo Fisher Scientific). Visual acquisition of the DNA fibers was done with a Zeiss Cell Observer fluorescent microscope equipped with a 40× NA 1.3 oil immersion objective and ZEN imaging software. Images were analyzed with the image processing program FIJI software (36 (link)).
5
DNA Fiber Analysis Protocol
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Cells were incubated as indicated with 100 μM CldU and 100 μM IdU. Cells were harvested and DNA fibers were obtained using the FiberPrep kit (Genomic Vision, EXT-001). DNA fibers were stretched on glass coverslips (Genomic Vision, COV-002-RUO) using the FiberComb Molecular Combing instrument (Genomic Vision, MCS-001). Slides were incubated with primary antibodies: Anti-BrdU BU1/75 (Abcam, 6326) for detection of CIdU; Anti-BrdU B44 (BD, 347580) for detection of IdU; Anti-single-stranded DNA (Millipore Sigma, MAB3034) for detection of DNA. Slides were washed with PBS, and incubated with secondary antibodies: Anti-mouse Cy3.5 preadsorbed (Abcam, 6946); Anti-rat Cy5 preadsorbed (Abcam, 6565); Anti-rabbit BV480-conjugated (BD Biosciences, 564879). Slides were mounted and imaged with a Leica SP5 confocal microscope and analyzed using LASX 3.3.0.16799 software.
6
Immunofluorescence Staining of DNA Damage Foci
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Cells were fixed in 3% formaldehyde, air dried and washed twice in PBS, then blocked using goat serum. Sections were then incubated O/N at 4C with the primary antibodies. Sections were washed three times in PBS prior to incubation with secondary antibodies for 30 minutes at RT. An anti γH2AX (S139) antibody (JBC301, EMD Millipore, MA) with secondary Alexa-Fluor 594 anti-mouse (A11032; Invitrogen) was used to stain foci. Immunofluorescence was observed at X100 or X60 magnification using NIKON 90i fluorescence microscope (photometric cooled mono CCD camera; Nikon), and foci were counted from at least 50 cells. Data are the mean of three experimental repeats. Anti laminB1 (clone B10, Santa Cruz Biotechnology); anti RAD51 (clone EPR4030(3), Abcam); anti MT1-MMP (clone EP1264Y, Abcam); anti-BrdU (B44, BD Biosciences), anti FAK (clone 2C5B9, Proteintech) were detected with FITC or Alexa-Fluor 594 conjugated secondary antibodies. All slides were mounted with Vectashield containing DAPI (Vector Laboratories).
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