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REACTIVITY SENSITIVITY MW (kDa) Isotype
All Endogenous Mouse IgG1
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Immunohistochemistry (Paraffin)

Immunohistochemical analysis of paraffin-embedded HeLa cells, without (left) and with (right) BrdU incorporation, using BrdU (Bu20a) Mouse mAb.

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Immunofluorescence (Immunocytochemistry)

Confocal immunofluorescent analysis of HeLa cells using BrdU (Bu20a) Mouse mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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Flow Cytometry

Flow cytometric analysis of Jurkat cells incorporated with BrdU (30 minutes), using BrdU (Bu20a) Mouse mAb versus propidium iodide (DNA content).

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Flow Cytometry

Flow cytometric analysis of Jurkat cells, untreated (blue) or BrdU incorporated for 30 minutes (green), using BrdU (Bu20a) Mouse mAb.

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Immunohistochemistry (Paraffin)

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. Xylene.
  2. Ethanol, anhydrous denatured, histological grade (100% and 95%).
  3. Deionized water (dH2O).
  4. Hematoxylin (optional).
  5. Wash Buffer:
    1. 1X Tris Buffered Saline with Tween® 20 (TBST): To prepare 1L 1X TBST add 100 ml 10X Tris Buffered Saline with Tween® 20 (#9997) to 900 ml dH20, mix.
  6. SignalStain® Antibody Diluent (#8112).
  7. Antigen Unmasking Citrate: 10 mM Sodium Citrate Buffer: To prepare 1 L, add 2.94 g sodium citrate trisodium salt dihydrate (C6H5Na3O7•2H2O) to 1 L dH2O. Adjust pH to 6.0.
  8. 3% Hydrogen Peroxide: To prepare 100 ml, add 10 ml 30% H2O2 to 90 ml dH2O.
  9. Blocking Solution: TBST/5% Normal Goat Serum: to 5 ml 1X TBST, add 250 µl Normal Goat Serum (#5425).
  10. Detection System: SignalStain® Boost IHC Detection Reagents (HRP, Mouse #8125).
  11. Substrate: Vector® NovaRED™ (Vector Laboratories).

B. Deparaffinization/Rehydration

NOTE: Do not allow slides to dry at any time during this procedure.

  1. Deparaffinize/hydrate sections:
    1. Incubate sections in three washes of xylene for 5 min each.
    2. Incubate sections in two washes of 100% ethanol for 10 min each.
    3. Incubate sections in two washes of 95% ethanol for 10 min each.
  2. Wash sections two times in dH2O for 5 min each.

C. Antigen Unmasking

For Citrate: Bring slides to a boil in 10 mM sodium citrate buffer, pH 6.0; maintain at a sub-boiling temperature for 10 min. Cool slides on bench top for 30 min.

D. Staining

  1. Wash sections in dH2O three times for 5 min each.
  2. Incubate sections in 3% hydrogen peroxide for 10 min.
  3. Wash sections in dH2O two times for 5 min each.
  4. Wash sections in wash buffer for 5 min.
  5. Block each section with 100–400 µl blocking solution for 1 hr at room temperature.
  6. Remove blocking solution and add 100–400 µl primary antibody diluted in SignalStain® Antibody Diluent (#8112) to each section. Incubate overnight at 4°C.
  7. Equilibrate SignalStain® Boost Detection Reagent (HRP, Mouse #8125) to room temperature.
  8. Remove antibody solution and wash sections with wash buffer three times for 5 min each.
  9. Cover section with 1–3 drops SignalStain® Boost Detection Reagent (HRP, Mouse #8125) as needed. Incubate in a humidified chamber for 30 min at room temperature.
  10. Wash sections three times with wash buffer for 5 min each.
  11. Prepare Vector® NovaRED™ per manufacturer's recommendations.
  12. Apply 100-400 µl substrate to each section and monitor closely. 5-15 minutes generally provides an acceptable staining intensity.
  13. Immerse slides in dH2O.
  14. If desired, counterstain sections with hematoxylin per manufacturer’s instructions.
  15. Wash sections in dH2O two times for 5 min each.
  16. Dehydrate sections:
    1. Incubate sections in 95% ethanol two times for 10 sec each.
    2. Repeat in 100% ethanol, incubating sections two times for 10 sec each.
    3. Repeat in xylene, incubating sections two times for 10 sec each.
  17. Mount sections with coverslips.

posted February 2010

revised November 2013

protocol id: 287

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Immunofluorescence (Immunocytochemistry)

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. Adjust pH to 8.0.
  2. BrdU (5-Bromo-2′-deoxyuridine) EMD biosciences (Cat. #203806)
  3. Ethanol, denatured 70%, Solution, American Bioanalytical (Cat. #CU00844)
  4. 1.5 M Hydrochloric acid
  5. Blocking Buffer (1X PBS / 5% normal serum):
    To prepare 10 ml, add 0.5 ml normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum (#5425)) and 0.5 mL 20X PBS to 9.0 mL dH2O, mix well.
  6. Antibody Dilution Buffer (1X PBS / 1% BSA): Add 0.1 g BSA (#9998) to 10 ml 1X PBS and mix well.
  7. Recommended Fluorochrome-conjugated Anti-Mouse secondary antibodies:

  8. Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).

B. Specimen Preparation - Cultured Cell Lines (IF-IC)

NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.

BrdU Incorporation: Dilute BrdU in fresh, pre-warmed growth medium to a final concentration of 0.03 mg/mL. Add mixture to cells and incubate at 37°C for 30 minutes.

  1. Aspirate media, cover cells completely with cold 70% ethanol.
  2. Allow cells to fix for 5 minutes at room temperature.
  3. Aspirate fixative, rinse three times in 1X PBS for 5 minutes each.
  4. Add 1.5M HCl and incubate for 30 minutes at room temperature.
  5. Aspirate HCl and rinse two times in 1X PBS for 5 minutes each.
  6. Proceed with Immunostaining section C.

C. Immunostaining

NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.

  1. Block specimen in Blocking Buffer for 60 minutes.
  2. While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer.
  3. Aspirate blocking solution, apply diluted primary antibody.
  4. Incubate overnight at 4°C.
  5. Rinse three times in 1X PBS for 5 minutes each.
  6. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1–2 hours at room temperature in dark.
  7. Rinse in 1X PBS as in step 5.
  8. Coverslip slides with Prolong® Gold Antifade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961) or apply just enough to cover cells in multiwell plate.
  9. For best results examine specimens immediately using appropriate excitation wavelength. For long term storage, store slides flat at 4°C protected from light.

posted November 2009

revised December 2010

protocol id: 33

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Flow Cytometry

A. Solutions and Reagents

  1. 1X Phosphate Buffered Saline (PBS): Dissolve 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4 and 0.24 g KH2PO4 in 800 mL distilled water (dH2O). Adjust the pH to 7.4 with HCl and the volume to 1 liter. Store at room temperature.
  2. BrdU (5-Bromo-2′-deoxyuridine) EMD biosciences (Cat. #203806)
  3. Ethanol, anhydrous denatured, histological grade, 100% and 95%
  4. 1.5 M Hydrochloric acid
  5. Incubation Buffer: Dissolve 0.5 g bovine serum albumin (BSA) in 100 mL 1X PBS. Store at 4°C.
  6. Recommended Anti-Mouse secondary antibodies::

B. BrdU Incorporation and Specimen Preparation

  1. Add BrdU to fresh, warm media for a final concentration of 0.03 mg/mL. Set aside.
  2. Collect ~50 million cells in tube by centrifugation and aspirate supernatant.
  3. Add 2 ml of BrdU-containing media to cell pellet, vortex, and incubate at 37°C for 30 minutes.
  4. Pellet cells by centrifugation and aspirate media.
  5. Add 2 ml of cold, 70% ethanol to cell pellet and mix.
  6. Allow cells to fix for 5 minutes at room temperature.
  7. Add 2–3 ml PBS and rinse by centrifugation three times.
  8. Add 1.5 M HCL and incubate for 30 minutes at room temperature.
  9. Add 2–3 ml PBS and rinse by centrifugation two times.
  10. Proceed with Immunostaining section C.

C. Immunostaining

NOTE: Account for isotype matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies. Count cells using a hemacytometer or alternative method.

  1. Aliquot 0.5–1 x 106 cells into each assay tube (by volume).
  2. Add 2–3 ml Incubation Buffer to each tube and rinse by centrifugation. Repeat.
  3. Resuspend cells in 100 µl Incubation Buffer per assay tube.
  4. Block in Incubation Buffer for 10 minutes at room temperature.
  5. Add the unconjugated primary antibody at the appropriate dilution to the assay tubes (see antibody data sheet for the appropriate dilution).
  6. Incubate for 1 hour at room temperature.
  7. Rinse as before in Incubation Buffer by centrifugation.
  8. Resuspend cells in fluorochrome-conjugated secondary antibody diluted in Incubation Buffer at the recommended dilution.
  9. Incubate for 30 minutes at room temperature.
  10. Rinse as before in Incubation Buffer by centrifugation.
  11. Resuspend cells in 0.5 ml PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to step D1.

D. Optional DNA Stain

  1. Resuspend cells in 0.5 ml of DNA dye (eg. DRAQ5® #4084).
  2. Incubate for at least 5 mins at room temperature.
  3. Analyze cells in DNA stain on flow cytometer.

posted December 2009

protocol id: 30

Product Usage Information

Application Dilutions
Immunohistochemistry 1:200
Immunofluorescence 1:1400
Flow Cytometry 1:200

Storage: Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Specificity / Sensitivity

BrdU (Bu20a) Mouse mAb detects BrdU when incorporated into single stranded DNA. DNA must be denatured for the epitope to be exposed and recognized by the antibody.


Species Reactivity: All Species Expected

Source / Purification

Monoclonal antibody is produced by immunizing animals with BrdU conjugated to BSA.

Halogenated nucleotides such as the pyrimidine analog bromodeoxyuridine (BrdU) are useful for labeling nascent DNA in living cells and tissues. BrdU becomes incorporated into replicating DNA in place of thymidine and subsequent immunodetection of BrdU using specific monoclonal antibodies allows labeling of cells in S phase of the cell cycle. After pulse-labeling cells or tissues with bromodeoxyuridine, BrdU (Bu20a) Mouse mAb can be used to detect BrdU incorporated into single stranded DNA. Please see our detailed protocol for information regarding the labeling procedure and denaturation of double stranded DNA for various immunodetection applications (1-4).


1.  Darzynkiewicz, Z. and Juan, G. (2001) Curr Protoc Cytom Chapter 7, Unit 7.7.

2.  Leif, R.C. et al. (2004) Cytometry A 58, 45-52.

3.  Staszkiewicz, J. et al. (2009) Biochem Biophys Res Commun 378, 539-44.

4.  Rothaeusler, K. and Baumgarth, N. (2007) Curr Protoc Cytom Chapter 7, Unit7.31.



For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.
DRAQ5® is a registered trademark of Biostatus Limited.