SpecificityThis antibody reacts with BrdU in single stranded DNA, BrdU attached to a protein carrier or free BrdU. It detects nucleated cells in S-Phase which have had BrdU incorporated into their DNA. Also reacts with chlorodeoxyuridine but with reduced staining. The antibody does not react with thymidine.
The antibody does not cross react with IdU.
The details of the immunogen for this antibody are not available.
General notesThe antibody recognises single stranded DNA so the DNA needs to be unraveled first. This can be done with DNAse, although this doesn't give the best results. Depending on the assay, acid denaturation with 2M HCL or heat denaturation are the most successful. Please note this step is critical in any assay with this antibody and is the area that should be modified to optimise results.
Detailed BrdU protocol is available in "Neuroscience protocols" on our "Protocol and troubleshooting tips" webpage (www.abcam.com/protocols).
Storage instructionsShipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
IHC (PFA fixed)
Use at an assay dependent concentration. PubMed: 26192438
Use at an assay dependent concentration.
1/40 - 1/200. PubMed: 16670699In addition, found to work at 1/400. For PFA fixed tissue use at 1/40, from PMID 16373695.
1/25 - 1/200.
ab18450 - Rat monoclonal IgG2a, is suitable for use as an isotype control with this antibody.
Use at an assay dependent concentration.
RelevanceThe immunocytochemical detection of bromodeoxyuridine (BrdU) incorporated into DNA is a powerful tool to study the cytokinetics of normal and neoplastic cells. In vitro or in vivo labeling of tumor cells with the thymidine analogue BrdU and the subsequent detection of incorporated BrdU with specific anti-BrdU monoclonal antibodies is an accurate and comprehensive method to quantitate the degree of DNA-synthesis.
BrdU is incorporated into the newly synthezised DNA of S-phase cells may provide an estimate for the fraction of cells in S-phase. Also dynamic proliferative information such as the S-phase transit rate and the potential doubling time can be obtained, by means of bivariate BrdU/DNA flow cytometric analysis.
ICC/IF image of ab6326 stained HeLa cells, both BrdU treated (left image) and normal cells (right image). The cells were 100% methanol fixed (5 min) and then subjected to acid hydrolysis using 2M HCL in 0.1% PBS-Tween for 30 minutes at room temperature to denature the DNA. They were then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab6326, 10µg/ml) overnight at +4°C. The secondary antibody (green) was ab98420, DyLight® 488 goat anti-rat IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. Positive staining can be seen in the BrdU treated cells, but not in the normal cells, demonstrating specificity for BrdU.
Flow Cytometry - Anti-BrdU antibody [BU1/75 (ICR1)] (ab6326)This image is courtesy of an anonymous Abreview
ab6326 staining BrdU in HeLa cells by Flow Cytometry. Cells were incubated with 10 µM BrdU for 30 minutes prior to being harvested with 1X trypsin-EDTA, washed twice in PBS containing 1% BSA, and fixed in 70% ethanol (added drop-wise) for at least 30 minutes on ice. Once fixed, pellets were acid denatured with HCl/Triton X-100 for 30 minutes at room temperature and then neutralised with sodium tetraborate. Pelleted cells were re-suspended in Tween/BSA/PBS to which primary antibody was then added (0.1 µg in 0.5% Tween 20 (v/v) plus 1% BSA in PBSA) and incubated for 30 minutes at room temperature. Secondary Alexa Fluor®488-conjugated Goat anti-Rat IgG (H+L) was used at 1/500 and incubated for 30 minutes at room temperature in the dark. Cells were pelleted once more and resuspended in PBS containing 5 µg/mL propidium iodide. Gating Strategy: Based on forward and side scatter, cells were gated into the region used for analysis. This was done by applying a large circle to a
Immunohistochemistry (PFA perfusion fixed frozen sections) - Anti-BrdU antibody [BU1/75 (ICR1)] (ab6326)This image is courtesy of an Abreview submitted by Musaad Alshammari.
ab6326 staining BrdU in mouse brain (dentate gyrus) tissue sections by Immunohistochemistry (PFA perfusion fixed frozen sections). Tissue samples were perfused with 1X PBS followed by 4% paraformaldehyde and then cryopreserved in 20-30% sucrose. 20-25 µm sections were permeablized with 1% Triton X-100 + 0.5% Tween 20 in 1X PBS. Sections were treated with 1 N HCL for 10 min followed by 2 N HCL for 10 min at RT and then 20 min at 37°C. Then sections were incubated with borate for pH correction and permeablized with 1 X TBS and blocked with 3-5% donkey serum. Samples were incubated with the primary antibody (1/1000 in 1X PBS + 0.1% Tween 20) at 4°C overnight. ab175475, an Alexa Fluor® 568-conjugated donkey anti-rabbit IgG (H+L) polyclonal (1/250) was used as the secondary antibody.
Immunocytochemistry - Anti-BrdU antibody [BU1/75 (ICR1)] (ab6326)This image is courtesy of an anonymous Abreview
ab6326 staining cultured cells of rat brain tissue by ICC. The sample was PFA fixed and permeabilized in 1M HCl prior to blocking with 5% serum for 1 hour at 25°C. The primary antibody was diluted 1/500 and incubated with the sample for 16 hours at 25°C. A biotinylated rabbit anti-rat IgG antibody, diluted 1/200, was used as the secondary.
Immunohistochemistry (Frozen sections) - Anti-BrdU antibody [BU1/75 (ICR1)] (ab6326)This image is courtesy of an anonymous Abreview
ab6326 staining BrdU in mouse brain tissue sections by IHC-Fr (paraformaldehyde-fixed frozen sections). Tissue samples were fixed with paraformaldehyde; permeabilized win 0.3% Triton X-100 and blocked with 5% Serum for 2 hours at 4°C. Before permeabilization samples were pretreated with 2N HCl at RT for 30 min and washed 3 times. The sample was incubated with primary antibody (1/200) at 4°C for 12 hours. An Alexa Fluor® 488-conjugated Goat polyclonal to rat IgG (1/500) was used as secondary antibody. BrdU staining shown in green and NewN staining showin in red.