Hot Summer. Cool Savings. | Start Saving >>
5292
BrdU (Bu20a) Mouse mAb

BrdU (Bu20a) Mouse mAb #5292

APPLICATIONS

REACTIVITY SENSITIVITY MW (kDa) Isotype
All Endogenous Mouse IgG1
IHC-P (paraffin)

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

Learn more about how we get our images
IF-IC

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

Learn more about how we get our images
Flow Cytometry

Flow cytometric analysis of Jurkat cells incorporated with BrdU (30 minutes), using BrdU (Bu20a) Mouse mAb versus DRAQ5® (DNA content).

Learn more about how we get our images
Flow Cytometry

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

Learn more about how we get our images
Flow Cytometry

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

Learn more about how we get our images

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. 1X Citrate Unmasking Solution: To prepare 250 mL of 1X citrate unmasking solution, dilute 25 ml of SignalStain® Citrate Unmasking Solution (10X) (#14746) with 225 mL of dH2O.
  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 or 1X Animal-Free Blocking Solution.
    1. TBST/5% Normal Goat Serum: to 5 ml 1X TBST, add 250 µl Normal Goat Serum (#5425).
    2. 1X Animal-Free Blocking Solution: to 4 mL of dH2O add 1 ml of Animal-Free Blocking Solution (5X) (#15019).
  10. Detection System: SignalStain® Boost IHC Detection Reagents (HRP, Mouse #8125).
  11. Substrate: SignalStain® DAB Substrate Kit (#8059).
  12. Hematoxylin: Hematoxylin (#14166).
  13. Mounting Medium: SignalStain® Mounting Medium (#14177).

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: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at a sub-boiling temperature (95°-98°C). 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 of preferred 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. Add 1 drop (30 µl) SignalStain® DAB Chromogen Concentrate to 1 ml SignalStain® DAB Diluent and mix well before use.
  12. Apply 100–400 µl SignalStain® DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.
  13. Immerse slides in dH2O.
  14. If desired, counterstain sections with hematoxylin (#14166).
  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 and mounting medium (#14177).

posted February 2010

revised March 2016

Protocol Id: 280

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

Flow Cytometry, Methanol Permeabilization Protocol for Mouse Antibodies

A. Solutions and Reagents

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

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 16% Formaldehyde (methanol free).
  3. 100% methanol.
  4. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.
  5. Recommended Anti-Mouse secondary antibodies::

B. Fixation

NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.

  1. Collect cells by centrifugation and aspirate supernatant.
  2. Resuspend cells in 0.5-1 ml 1X PBS. Add formaldehyde to obtain a final concentration of 4%.
  3. Fix for 15 min at room temperature.
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS.

C. Permeabilization

  1. Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol.
  2. Incubate 30 min on ice.
  3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

NOTE: Count cells using a hemocytometer or alternative method.

  1. Aliquot desired number of cells into tubes or wells.
  2. Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard supernatant in appropriate waste container. Repeat if necessary.
  3. Resuspend cells in 100 µl of diluted primary antibody (prepared in incubation buffer at the recommended dilution).
  4. Incubate for 1 hr at room temperature.
  5. Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
  6. Resuspend cells in 100 µl of diluted fluorochrome-conjugated secondary antibody (prepared in incubation buffer at recommended dilution).
  7. Incubate for 30 min at room temperature.
  8. Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
  9. Resuspend cells in 1X PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA stain (Section E).

E. Optional DNA Dye

  1. Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
  2. Incubate for at least 5 min at room temperature.
  3. Analyze cells in DNA staining solution on flow cytometer.

posted June 2005

revised June 2017

Protocol Id: 406

Application Dilutions
Immunohistochemistry (Paraffin) 1:200
Immunofluorescence (Immunocytochemistry) 1:1000
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.

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

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.

News from the Bench

Discover what’s going on at CST, receive our latest application notes and tips, read our science features, and learn about our products.

Subscribe