Analysis Note

ControlAfter incorporation of BrdU, all DNA containing species

Application

Use Anti-BrdU Antibody, clone AH4H7-1 / 131-14871 (Mouse Monoclonal Antibody) validated in FC, ICC, IHC to detect Bromodeoxyuridine also known as BrdU.

Immunohistochemistry: (6 µg/ml)

Flow Cytometry: (0.2 µg/100 µl/10E6 cells) Optimal working dilutions must be determined by end user.

APPLICATIONS

Flow cytometry:The method below is based on that of M. Vanderlaan et al. (1986). Variations of this method exist in the literature, one consideration being the effect various fixation procedures have on the light-scattering properties of different cell populations. Procedure:

1. To label cells, pulse with 10 µM bromodeoxyuridine for 30 minutes. Harvest cells from culture.

2. Fix cells in 70% ethanol at +2-8°C for at least 30 min. Extract histones by resuspending cells in 1 mL chilled 0.1 M HCI containing 0.5% Triton X-100; incubate the suspension on ice for 10 minutes. Dilute acid with 5 mL distilled water and centrifuge at 200 x g for 10 min. Resuspend cells in 2 mL distilled water.

3. Denature cellular DNA by submerging the cell suspension into a boiling water bath for 10 min. Afterwards, quickly cool by placing the cell suspension in an ice slurry for several minutes. Wash cells in PBS that contains 0.5% Triton X-100.

4. Resuspend the cells (1-2 x 10 6 cells) in 100 µL of solution containing approximately 2 µg/mL anti-bromodeoxyuridine antibody diluted in PBS containing 0.1% BSA (0.2 µg/test). Incubate for 30 min at room temperature. Wash cells with PBS.

5. Resuspend cells in 100 µL of diluted goat anti-mouse IgG-FlTC Wash cells with PBS.

APPLICATIONS (Cont.)

Immunohistochemistry: Below is a procedure for staining cells that have been labeled with BrdU in vivo or in vitro. The procedure is based on the methods of B. Schutte et al. (1987) and D. Campana et al. (1988).

Preparation of tissue:

Inject animal with 50 mg BrdU/kg body weight. Sacrifice animal one hour later and remove organ or tissue under study. Embed tissue in OCT medium and snap-freeze by immersion into liquid nitrogen.Cut 4 mm frozen sections with a cryostat. Place sections on either albumin- or gelatin-coated slides.

Preparation of cells:

Pulse cells with 10 mM BrdU for 60 min. Cells grown on coverslips, or cytocentrifuge preparations made from cells grown in suspension, can be used for anti-bromodeoxyuridine staining according to the procedure below.

Procedure

1. Fix tissue sections or cells (on slide or coverglass) by immersing in absolute methanol for 10 minutes at +2-8°C. Air dry after removing from fixative. The slides can be stored at -20°C in a sealed box, or rehydrated to prepare for the assay procedure. To rehydrate, immerse in PBS for 3 min.

2. Denature DNA by incubating the slides in 2 N HCI for 60 min at +37°C.

3. Neutralize the acid by immersing the slides in 0.1 M borate buffer, pH 8.5. Change the buffer twice over a 10 min period.

4. Wash slides with PBS, changing the solution three times over a 10 min period.

5. Place slides in a humidified chamber (e.g., a sealed plastic box layered with wet paper towels) and cover cells with 150-300 µL of solution containing approximately 6 µg/mL anti-bromodeoxyuridine antibody diluted in PBS with 0.1% BSA. Incubate for 60 min at room temperature.

6. Wash slides with PBS, changing the solution three times over a 10 min period.

7. Apply optimal dilution of a second antibody conjugate (e.g., anti-mouse IgG-peroxidase), incubate, wash, and perform detection with a substrate that produces an insoluble product. After detection, counterstain with Harris-modified hematoxylin if desired. Slides can then be dehydrated and mounted.

Research CategoryEpigenetics & Nuclear Function

Research Sub CategoryCell Cycle, DNA Replication & Repair

Disclaimer

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

General description

Bromodeoxyuridine (BrdU) is a thymidine analog and is specifically incor-porated into DNA during DNA synthesis. Anti-bromodeoxyuridine monoclonal antibody is used to identify cells that have incorporated BrdU. This immunological detection scheme has several advantages over the use of radioactive thymidine incorporation for identifying cells under-going replication. Labeling and detection can be performed the same day instead of waiting several days, as required for autoradiography of tritium-labeled cells, and the necessity of using multiple specimens for obtaining the optimal exposure time is eliminated. In addition, anti-bromodeoxyuridine staining with flow cytometric analysis allows multiple parameters to be evaluated simultaneously. Anti-bromodeoxyuridine monoclonal antibody has been used for identi-fying proliferating cells in blood (Campana et al., 1988), tissues (Schutte et al., 1987; Hayashi, et al., 1988), tumors (Hoshino et al., 1986; Morstyn et al., 1983), as well as for determining plasma cell labeling indices (Greipp et al., 1985).

Immunogen

Bromodeoxyuridine-bovine serum albumin concentrate

Legal Information

CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany

Linkage

Replaces: MAB1467

Other Notes

Concentration: Please refer to the Certificate of Analysis for the lot-specific concentration.

Physical form

Protein A purified

Liquid in 10 mM Phosphate buffer, pH 7.4 containing 150 mM NaCl and 0.1% sodium azide

Format: Purified

Specificity

Binds to bromodeoxyuridine and crossreacts with iodouridine (10%). Anti-bromodeoxyuridine does not crossreact with fluorodeoxyuridine, nor with any endogenous cellular components such as thymidine or uridine.

Storage and Stability

Maintain for 6 months at -20°C from date of shipment. Aliquot to avoid repeated freezing and thawing. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.

Target description

dependent upon the molecular weight of the bromodeoxyuridine incorporated protein being detected