BrdU

IgG

N/A

1. 93 mg/ml Storage

ELISA: Use at an assay dependent dilution. IHC-P: Use at a dilution of 1/10. IHC-Fr: Use at a concentration of 10 µ g/ml. IP: Use at a concentration of 25 - 100 µ g/ml. Fixation in cold methanol for 30 minutes followed by immersion in 7 x 10^-3 M NaOH for 10-15 seconds allows BrdU staining with the simultaneous detection of nuclear cytoplasmic and membrane assigns as well as preservation of morphological detail. The unconjugated antibody was tested using immunoprecipitation against 5-Methyl Cytosine (5-MeC) and bromodeoxyuridine (BrdU) or control (no antigen) and assayed by A-405 spectrophotometry. At a concentration of 25 µ g/mL this product demonstrates 8 fold higher reactivity against BrdU than 5-MeC. Product titers out to 50 ng/mL in the purified form. For best results on unconjugated antibody use the product at a concentration of 25 to 100ug/mL. Nearly complete immunoprecipitation was obtained at concentrations of 100 to 500ug/mL. The product has also been tested for utility for staining of bromodeoxyuridine incorporated into DNA of replicating cells. When utilized at a purified concentration of 10 µ g/mL the product is comparable to commercially available monoclonal antibodies commonly used for the same purpose. A key issue is that it is necessary to denature the DNA so as to expose the base to the antibodies. One protocol is: Sachiko Matsuuraa and Kazuo Suzukia Immunohistochemical Analysis of DNA Synthesis During Chronic Stimulation with Isoproterenol in Mouse Submandibular Gland . Journal of Histochemistry and Cytochemistry Vol. 45 1137-1146 Immunohistochemistry DNA-synthesizing cells were detected by peroxidase-anti-peroxidase (PAP) immunostaining with anti-BrdU antibodies by the method of Harms et al. 1986 which was slightly modified as follows. Sections were treated with 2 N HCl at 60C for 15 min and washed in running water for 15 min. After dehydration in an ethanol series the sections were etched with xylene (15 min twice). Nuclear proteins which mask incorporated BrdU were digested with 0. 025% protease (Type V; Sigma) in PBS at 37C for 10 min and the sections were then washed with cold PBS three times for 5 min. Not tested in other applications. Optimal dilutions/concentrations should be determined by the end user. The 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 the S-phase cells and can thus 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. N/A