Bromodeoxyuridine (BrDU) antibody

Details for Product No. ABIN152983
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Antigen
Reactivity
Chemical
(168), (13), (5), (5), (4), (2), (1), (1)
Host
Rat
(127), (30), (26), (14), (1)
Clonality (Clone)
Monoclonal ()
Conjugate
Un-conjugated
(24), (12), (8), (4), (3), (3), (3), (3), (3), (3), (3), (3), (1), (1), (1)
Application
Flow Cytometry (FACS), Immunocytochemistry (ICC), Immunofluorescence (IF), Immunohistochemistry (IHC), Immunohistochemistry (Frozen Sections) (IHC (fro)), Immunohistochemistry (Paraffin-embedded Sections) (IHC (p))
(100), (84), (47), (41), (36), (33), (30), (26), (18), (12), (10), (3), (1), (1), (1), (1)
Pubmed 5 references available
Quantity 0.1 mg
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Catalog No. ABIN152983
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Immunogen Made to Chemical BrdU
Clone BU1-75 (ICR1)
Isotype IgG2a
Specificity This 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 weakly with chlorodeoxyuridine, but does not cross react with thymidine or iododeoxyuridine.
Purification Protein G purified
Background The immunocytochemical detection of bromodeoxyuridine (BrdU) incorporated into DNAis a powerful tool to study the cytokinetics of normal and neoplastic cells. In vitro or invivo labeling of cells with the thymidine analogue BrdU and the subsequent detection ofincorporated BrdU with specific anti-BrdU monoclonal antibodies is an accurate andcomprehensive method to quantitate the degree of DNA-synthesis. BrdU is incorporated into the newly synthezised DNA of the S-phase cells and can thusprovide an estimate for the fraction of cells in S-phase. Also dynamic proliferativeinformation (such as the S-phase transit rate and the potential doubling time) can beobtained, by means of bivariate BrdU/DNA flow cytometric analysis.
Research Area Proliferation Markers
Application Notes Clone BU1/75 (ICR1) can be used for labeling paraffin-embedded tissue sections fixed in formalin. Denaturation of the DNA is critical for successful staining of BrdU. This can be achieved by exposing cells to heat, or acid. For heat-induced epitope retrieval, 10mM citrate buffer pH6.0 is recommended. Alternatively, a 30 min incubation in 2M HCl can be performed. The HCl must then be neutralized for 2 min with 0.1 M Na2B4O7. Pretreatment of tissues with proteinase K should be avoided. Immunohistochemistry-Frozen was reported in scientific literature.
Recommended dilutions: Flow Cytometry 1:25-1:200, Immunocytochemistry/Immunofluorescence 1:10-1:500, Immunohistochemistry 1:10-1:500, Immunohistochemistry-Frozen 1:10-1:500, Immunohistochemistry-Paraffin 1:25-1:200
Protocol Protocol specific for BrdU Antibody Flow Cytometry Analysis: Prepare the following solutions before proceeding:Phosphate buffered saline (PBS)2N HCl, 0.5 % Triton X-100PBS containing 0.05 % Tween-20PBS containing 1 % BSA (PBS/BSA)10mg/ml Propidium iodide (PI)As a positive control, BrdU labeled cells maybe obtained from Phoenix Flow Systems (http://www.phnxflow.com), catalogue number ACNC12.
1. Add BrdU to the cell suspension in culture medium to a final concentration of 10 umol/L and incubate for 30 minutes in a CO2 incubator at 37C.
. Wash cells twice with PBS/BSA, and resuspend in PBS
. Add cells slowly into 5ml of 70 % ethanol at -20C, mixing continuously (vortex preferred). Incubate on ice for 30 minutes.
. Centrifuge at 500g for 10 minutes, decant supernatant, and resuspend cell pellet.
. Add 2ml 2N HCl, 0.5 % Triton X-100 and incubate the cells for 30 minutes at room temperature (preferably on a rocking platform).
. Centrifuge at 500g for 10 minutes, decant supernatant and resuspend in 3 mL 0.1M Na2B4O7, pH 8.
. Centrifuge at 500g for 10 minutes, decant supernatant and resuspend the cells in PBS/BSA + 0.05 % Tween
. Adjust cell concentration to 1 x 10(7)/ml
. Aliquot 100ul of cell suspension into required number of 12 x 75mm tubes.
. Incubate the cells with the anti-BrdU antibody at the recommended dilution for 30 minutes at room temperature.
. Add 2 mLs PBS/BSA and centrifuge the cells at 1000rpm for 5 minutes.
. If a secondary antibody layer is required then decant the wash and incubate the cells with the secondary antibody for 30 minutes at room temperature. If no secondary antibody layer is required then proceed to step 13.
. Wash the cells after the secondary antibody layer by repeating step 10.
. Decant off the wash and add 1ml PBS containing 10ug/ml PI (Dilute the 10mg/ml solution of PI 1/1000 in a suitable volume of PBS)
. Analyse cells by flow cytometry following the manufacturer's instructions. The PI should be read on the appropriate channel set to the Peak/Area and not log scale.IMMUNOHISTOLOGY Formalin-fixed paraffin-embedded tissue sections:Clone BU1/75 (ICR1) can be used for labelling paraffin-embedded tissue sections fixed in formalin. Pretreatment of tissues with heat-induced epitope retrieval using 10mM citrate buffer pH6.0 is recommended. Pretreatment of tissues with proteinase K should be avoided.
Restrictions For Research Use only
Concentration 1.0 mg/mL
Buffer PBS, Sodium Azide
Preservative Sodium azide
Precaution of Use WARNING: Reagents contain sodium azide. Sodium azide is very toxic if ingested or inhaled. Avoid contact with skin, eyes, or clothing. Wear eye or face protection when handling. If skin or eye contact occurs, wash with copious amounts of water. If ingested or inhaled, contact a physician immediately. Sodium azide yields toxic hydrazoic acid under acidic conditions. Dilute azide-containing compounds in running water before discarding to avoid accumulation of potentially explosive deposits in lead or copper plumbing.
Handling Advice Avoid freeze-thaw cycles
Storage 4 °C
Storage Comment 4 °C short term. Aliquot and store at -20 °C long term.
General Barbie, Kudlow, Frock et al.: "Nuclear reorganization of mammalian DNA synthesis prior to cell cycle exit." in: Molecular and cellular biology, Vol. 24, Issue 2, pp. 595-607, 2003 (PubMed).

Siegenthaler, Tremper-Wells, Miller: "Foxg1 haploinsufficiency reduces the population of cortical intermediate progenitor cells: effect of increased p21 expression." in: Cerebral cortex (New York, N.Y. : 1991), Vol. 18, Issue 8, pp. 1865-75, 2008 (PubMed).

Denis-Donini, Dellarole, Crociara et al.: "Impaired adult neurogenesis associated with short-term memory defects in NF-kappaB p50-deficient mice." in: The Journal of neuroscience : the official journal of the Society for Neuroscience, Vol. 28, Issue 15, pp. 3911-9, 2008 (PubMed).

Jiao, Feldheim, Chen: "Ephrins as negative regulators of adult neurogenesis in diverse regions of the central nervous system." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 105, Issue 25, pp. 8778-83, 2008 (PubMed).

Chang, Gaysinskaya, Karatayev et al.: "Maternal high-fat diet and fetal programming: increased proliferation of hypothalamic peptide-producing neurons that increase risk for overeating and obesity." in: The Journal of neuroscience : the official journal of the Society for Neuroscience, Vol. 28, Issue 46, pp. 12107-19, 2008 (PubMed).

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