Immunohistochemical analysis of paraffin-embedded HeLa cells, without (left) and with (right) BrdU incorporation, using BrdU (Bu20a) Mouse mAb.
Confocal immunofluorescent analysis of HeLa cells incorporated with BrdU (30 μg/mL, 30 min) using BrdU (Bu20a) Mouse mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Flow cytometric analysis of Jurkat cells incorporated with BrdU (30 minutes), using BrdU (Bu20a) Mouse mAb versus DRAQ5® (DNA content).
Flow cytometric analysis of Jurkat cells incorporated with BrdU (30 minutes), using BrdU (Bu20a) Mouse mAb versus propidium iodide (DNA content).
Flow cytometric analysis of Jurkat cells, untreated (blue) or BrdU incorporated for 30 minutes (green), using BrdU (Bu20a) Mouse mAb.
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.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: Do not allow slides to dry at any time during this procedure.
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.
|SignalStain® Boost IHC Detection Reagent (HRP, Mouse) #8125||SignalStain® Boost IHC Detection Reagent (AP, Mouse) #31926|
|SignalStain® DAB Substrate Kit #8059||SignalStain® Vibrant Red Alkaline Phosphatase Substrate Kit #76713|
|SignalStain® Vivid Purple Peroxidase Substrate Kit #96632|
NOTE: Use of detection reagents other than those specified in this protocol may require further optimization of the primary antibody to account for the different sensitivities of the detection reagents.
posted February 2010
revised June 2020
Protocol Id: 280
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
Recommended Fluorochrome-conjugated Anti-Mouse secondary antibodies:
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.
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.
posted November 2009
revised December 2010
Protocol Id: 33
NOTE: Account for isotype matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies. Count cells using a hemacytometer or alternative method.
posted December 2009
Protocol Id: 30
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.
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).
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