Chemicon Internation, Inc. - Product Data Sheet - Millipore

CATALOG NUMBER: MAB3424
LOT NUMBER:
QUANTITY: 50 μg
CONCENTRATION: 1.1 mg/mL
USA & Canada Phone: +1(800) 437-7500 Fax: +1 (951) 676-9209
Australia +61 3 9839 2000
www.millipore.com
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MOUSE ANTI-BROMODEOXYURIDINE (BrdU)
MONOCLONAL ANTIBODY
SPECIFICITY: The antibody specifically 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.
BACKGROUND: 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, antibromodeoxyuridine
staining with flow cytometric analysis allows multiple parameters to be
evaluated simultaneously. Anti-bromodeoxyuridine monoclonal antibody has been used for
identifying proliferating cells in blood (1), tissues (2,3), tumors (4–7), as well as for
determining plasma cell labeling indices (8).
IMMUNOGEN: Bromodeoxyuridine-bovine serum albumin concentrate
ISOTYPE: IgG1
CLONE: AH4H7-1 / 131-14871
APPLICATIONS: Immunohistochemistry: 6 μg/mL
Flow Cytometry: 0.2 μg/100 μL/10 6 cells
Optimal working dilutions must be determined by end user.
FORMAT: Protein A purified immunoglobulin.
PRESENTATION: Liquid in 10 mM Phosphate buffer, pH 7.4 containing 150 mM NaCl and 0.1% sodium azide.
STORAGE/HANDLING: Maintain frozen at -20°C in undiluted aliquots for up to 6 months after date of receipt. Avoid
repeated freeze/thaw cycles.
REFERENCES: 1. Demyanenko, G.P., et al., Neuron (2004) 44:423-437.
2. Emsley, J.G. and T. Hagg, (2003) Experimental Neurology 183:273-285.
3. Campana, D., Coustan-Smith, E. and Janossy, G. (1988) J. Immunol. Meth. 107:79.
4. Schutte, B., Reynders, M.M.J., Bosman, F.T. and Blijham, G.H. (1987)
J. Histochem. and Cytochem. 35:1343.
5. Hayashi, Y., Koike, M., Matsutani, M. and Hoshino, T. (1988) J. Histochem. and
Cytochem. 36:511.

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6. Hoshino, T., Nagashima, T., Cho, K., Murovic, J., Hodes, J., Wilson, C., Edwards, M. and
Pitts, L. (1986) Int. J. Cancer 38:369.
7. Nagashima, T., Murovic, J., Hoshino, T., Wilson, C., and Dearmond, S. (1986) J.
Neurosurg. 64:588.
8. Nagashima, T., DeArmond, S., Murovic, J. and Hoshino, T. (1985) Acta Neuropathol.
67:155.
9. Morstyn, G., Hsu, S.-M., Kinsella, T., Gratzner, H., Russo, A. and Mitchell, J. (1983) J.
Clinical Invest. 72:1844.
10. Greipp, P.R., Witzig, T.E. and Gonchoroff, N.J. (1985) Amer. J. Hematol. 20: 289.
11. Vanderlaan, M., Watkins, B., Thomas, C., Dolbeare, F., and Stanker, L. (1986)
Cytometry 7:499.
Flow cytometry:
APPLICATIONS
The method below is based on that of M. Vanderlaan et al. (9). 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.

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Australia +61 3 9839 2000
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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. (2) and D. Campana et al.
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. (Optimal concentration should be determined by user.)
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.
For research use only; not for use in diagnostic procedures.
Important Note: During shipment, small volumes of product will occasionally become entrapped in the seal of the
product vial. For products with volumes of 200 μL or less, we recommend gently tapping the vial on a
hard surface or briefly centrifuging the vial in a tabletop centrifuge to dislodge any liquid in the
container’s cap.
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