Anti-Bromodeoxyuridine-Fluorescein

For life science research only. Not for use in diagnostic procedures.

Mouse monoclonal antibody to bromodeoxyuridine (5-bromo-2’-deoxyuridine-5’-monophosphate) conjugated to fluorescein, solution, formalin grade 1 Cat. No. 11 202 693 001 50 ␮g (500 ␮l) y Version 08 Content version: March 2016 Store at Ϫ15 to Ϫ25°C

1. What this Product does 2.2 Procedures

Contents Flow cytometry 50 ␮g Anti-Bromodeoxyuridine-Fluorescein is supplied in 0.5 ml of The method below is based on that of M. Vanderlaan et al. (9). Varia- phosphate-buffered saline (PBS), pH 7.4, containing 0.09% (w/v) tions of this method exist in the literature, one consideration being the sodium azide and 0.2% (w/v) gelatin for stability. effect various fixation procedures have on the light-scattering proper- ties of different cell populations: Storage and Stability Step Action If stored at Ϫ15 to Ϫ25°C, the lyophilized and liquid antibody preparations are stable until the expiration date printed on the label. ³ To label cells, incubate with 10 ␮M 5-bromo-2'-deoxyuridine* for 30 min. Harvest cells from culture.  Avoid repeated freezing and thawing. ᕢ ϩ ϩ  The antibody is shipped at ambient temperature. Fix cells in 70% (v/v) ethanol at 2 to 8°C for at least 30 minutes. Application ᕣ Extract histones by resuspending cells in 1 ml chilled 0.1 M Cryosections, Immunochemistry, Flow cytometry, Paraffin sections. HCI containing 0.5% (v/v) Triton X-100; incubate the suspen- Anti-Bromodeoxyuridine-Fluorescein monoclonal antibody can be sion on ice for 10 min. used for identifying proliferating cells in blood (1), tissues (2,3), and Dilute acid with 5 ml redist. water and centrifuge at 200 ϫ g tumors (4–7), as well as for determining plasma cell labeling indices for 10 min. Resuspend cells in 2 ml distilled water. (8). The antibody may be applied for the qualitative measurement of ᕤ Denature cellular DNA by submerging the cell suspension cell proliferation (BrdU incorporation) on a single-cell level. into a boiling water bath for 10 min. Afterwards, quickly cool by placing the cell suspension in an Product Characteristics ice slurry for several minutes. Wash cells in PBS that contains 0.5% (v/v) Triton X-100. Specificity The antibody specifically binds to bromodeoxyuri- ᕥ Resuspend the cells (1 – 2 ϫ 106 cells) in 100 ␮l of solution dine and crossreacts with iodouridine (10%). Anti- containing approximately 5 ␮g/ml Anti-Bromodeoxyuridine- Bromodeoxyuridine-Fluorescein does not crossre- Fluorescein diluted in PBS containing 0.1% (w/v) BSA (0.5 ␮g act with fluorodeoxy-uridine, nor with any endoge- antibody/test). Incubate for 30 min at +15 to +25°C. Wash nous cellular components such as thymidine or cells with PBS. uridine. * available from Roche Diagnostics Clone BMC 9318

Subtype Mouse IgG1 Immunohistochemistry Purity The antibody is Ն90% pure as determined by SDS- Below is a procedure for staining cells that have been labeled with PAGE with Coomassie-blue staining, and by HPLC BrdU in vivo or in vitro. The procedure is based on the methods of B. Schutte et al. (2) and D. Campana et al. (1). 2. How to Use this Product Preparation of tissue 2.1 Before you begin Inject animal with 50 mg BrdU/kg body weight. Sacrifice animal 1 h later and remove organ or tissue under study. Embed tissue in OCT Working concentration medium and snap-freeze by immersion into liquid nitrogen. Cut 4 ␮m ␮ ␮ 6 frozen sections with a cryostat. Place sections on either albumin- or Use approximately 0.5 g/100 l/10 cells for flow cytometry and gelatin-coated slides. 50 ␮ g/ml for immunohistochemistry. These are guidelines only. The optimal concentration should be determined by the investigator. Dilu- Preparation of cells tions should be made in PBS (pH 7.4) containing 0.1% BSA to maintain ␮ stability of the antibody. Incubate cells with 10 M BrdU for 60-80 min, depending on the proliferation rate of the cells. Cells grown on coverslips, or cytocentrifuge preparations made from cells grown in suspension, can be used for Anti-Bromodeoxyuridine-Fluorescein staining according to the procedure below:

1 “During apoptosis, caspases cleave intracellular proteins; the corre- sponding caspase cleaving sites are formalin resistant. Formalin grade antibodies recognize cleavage sites in apoptotic cells that are not accessible in normal cells, which allows for determination of caspase activity, even in formalin-fixed, paraffin embedded tissue.” 0316.11274414001 sigma-aldrich.com 4. Supplementary Information Step Action ³ Fix tissue sections or cells (on slide or coverglass) by Changes to Previous Version immersing in absolute methanol for 10 minutes at ϩ2 to • Editorial changes ϩ 8°C. Air dry after removing from fixative. Text Conventions The slides can be stored at Ϫ15 to Ϫ25°C in a sealed box, or rehydrated to prepare for the assay procedure. To rehydrate, To make information consistent and understandable, the following text immerse in PBS for 3 min. conventions are used in this document: ᕢ Denature DNA by incubating the slides in 2 N HCI for 60 min at ϩ37°C. Text Convention Use ᕣ Numbered instructions Steps in a procedure that must be performed Neutralize the acid by immersing the slides in 0.1 M borate ᕡ ᕢ buffer, pH 8.5. Change the buffer twice over a 10 min period. labeled , etc. in the order listed. ᕤ Wash slides with PBS, changing the solution three times over Asterisk * Denotes a product available from Roche a 10 min period. Diagnostics. ᕥ Place slides in a humidified chamber (e.g., a sealed plastic Symbols box layered with wet paper towels) and cover cells with 100– Symbols are used in this document to highlight important information: 300 ␮l of solution containing approx. 50 ␮g/ml Anti-Bro- modeoxyuridine-Fluorescein diluted in PBS with 0.1% BSA. Incubate for 60 min at +15 to +25°C. Symbol Description  Protect cells from light. Information Note: ᕦ Wash slides with PBS, changing the solution three times over  Additional information about the current topic or proce- a 10 min period. dure.  After detection, counterstain with Harris-modified hematoxylin if Important Note: desired. Slides can then be dehydrated and mounted.  Information critical to the success of the procedure or use of the product. 3. Additional Information on this Product Background Information Trademarks Bromodeoxyuridine (BrdU) is a thymidine analog and is specifically All brands or product names are trademark of their respective holders. incorporated into DNA during DNA synthesis. Anti-Bromodeoxyuri- dine-Fluorescein is used to identify cells that have incorporated BrdU. Regulatory Disclaimer This immunological detection scheme has several advantages over the For life science research only. Not for use in diagnostic procedures. use of radioactive thymidine incorporation for identifying cells under- going replication. Labeling and detection can be performed the same Disclaimer of License day instead of waiting several days, as required for autoradiography of For patent license limitations for individual products please refer to: tritium-labeled cells, and the necessity of using multiple specimens for List of biochemical reagent products obtaining the optimal exposure time is eliminated. In addition, anti- bromodeoxyuridine staining with flow cytometric analysis allows multiple parameters to be evaluated simultaneously. Preparation BALB/c mice were immunized with a bromodeoxyuridine-bovine serum albumin conjugate. Lymphocytes isolated from the spleen were fused with Ag8.653 myeloma cells to create the BMC 9318 clone. The antibody was produced in ascites in BALB/c mice and purified by ion- exchange chromatography. Quality The titer of each lot of anti-bromodeoxyuridine is determined by immunohistochemistry as described above.

References 1 Campana, D., Coustan-Smith, E., and Janossy, G. (1988) J. Immunol. Meth. 107, 79. 2 Schutte, B., Reynders, M.M.J., Bosman, F.T., and Blijham, G.H. (1987) J. Histochem. and Cytochem. 35, 1343. 3 Hayashi, Y., Koike, M., Matsutani, M., and Hoshino, T. (1988) J. Histo- chem. and Cytochem. 36, 511. 4 Hoshino, T., Nagashima, T., Cho, K., Murovic, J., Hodes, J., Wilson, C., Edwards, M., and Pitts, L. (1986) Int. J. Cancer 38: 369. 5 Nagashima, T., Murovic, J., Hoshino, T., Wilson, C., and Dearmond, S. (1986) J. Neurosurg. 64, 588. Contact and Support 6 Nagashima, T., DeArmond, S., Murovic, J., and Hoshino, T. (1985) Acta To ask questions, solve problems, suggest enhancements and report new Neuropathol. 67, 155. applications, please visit our Online Technical Support Site. 7 Morstyn, G., Hsu, S.-M., Kinsella, T., Gratzner, H., Russo, A., and Mitchell, J. (1983) J. Clinical Invest. 72, 1844. To call, write, fax, or email us, visit sigma-aldrich.com, and select your home 8 Greipp, P.R., Witzig, T.E. and Gonchoroff, N.J. (1985) Amer. J. Hematol. country. Country-specific contact information will be displayed. 20, 289. 9 Vanderlaan, M., Watkins, B., Thomas, C., Dolbeare, F., and Stanker, L. (1986) Cytometry 7, 499.

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