BrdU Antibody Protocols

Anti-BrdU antibody

Staining protocols

We have tested and can confirm that the anti-BrdU antibody mouse monoclonal clone Bu20a and rat monoclonal clone BU1/75 (ICR1) are suitable for flow cytometry and immunohistochemistry on paraffin-embedded tissue sections fixed in formalin.The following methods were used and provide a useful guide for using anti-BrdU antibodies.

Preparation of reagents for staining

The following solutions should be prepared:

  • Phosphate buffered saline (PBS)
  • 2 N HCl containing 0.5% Triton X-100
  • PBS containing 0.05% Tween®-20
  • PBS containing 1% BSA (PBS/BSA)
  • 10 mg/ml propidium iodide (PI)
  • 0.1 M Na2B4O7, pH 8.5

Flow cytometry staining with anti-BrdU antibodies

HL-60 cells were pulse labeled with BrdU for 45 minutes prior to harvesting and then incubated with primary antibody Rat BrdU antibody

Fig 1. HL-60 cells were pulse labeled with BrdU for 45 minutes prior to harvesting and then incubated with primary antibody Rat anti-BrdU (MCA2060GA) clone BU1/75 diluted 1/100 followed by Rabbit anti-Rat IgG secondary antibody (STAR17B) FITC-conjugated, diluted 1/200

  • Add BrdU to the cell suspension in culture medium to a final concentration of 10 μM and incubate for 30 minutes in a CO2 incubator at 37°C.
  • Wash cells twice with PBS/BSA, by centrifuging at 500 x g for 10 minutes, decant supernatant and resuspend in a minimum volume of PBS.
  • Add cells slowly into 5 ml of 70% ethanol at -20°C, mixing continuously (vortex preferred). Incubate on ice for 30 minutes.
  • Centrifuge at 500 x g for 10 minutes, decant supernatant, and resuspend cell pellet.
  • Add 2 ml of 2 N HCl containing 0.5% Triton X-100, and incubate the cells for 30 minutes at room temperature (preferably on a rocking platform). Denaturation of the DNA is critical for successful staining of BrdU. This can be achieved by exposing cells to HCl.
  • Centrifuge at 500 x g for 10 minutes, decant supernatant, and resuspend in 3 ml of 0.1 M Na2B4O7, pH 8.5.
  • Centrifuge at 500 x g for 10 minutes, decant supernatant, and resuspend the cells in PBS/BSA + 0.05% Tween®-20. Adjust cell concentration to 1 × 107 cells/ml.
  • Aliquot 100 μl of the cell suspension into required number of FACS tubes.
  • Incubate the cells with anti-BrdU antibody (Bu20a or BU1/75) at the recommended dilution for 45 minutes at room temperature or overnight at 4oC.
  • Wash Step: Add 2 ml of PBS/BSA, and centrifuge the cells at 500 x g for 5 minutes.
  • If a secondary antibody is required, then decant the supernatant and incubate the cells with the secondary antibody for 30 minutes at room temperature (see individual datasheet for recommended dilution). Wash the cells by repeating wash step.
  • If no secondary antibody layer is required then proceed to next step.
  • Decant the supernatant and add 1 ml of PBS containing 10 μg/ml of PI.
  • Analyze cells by flow cytometry following the manufacturer’s instructions. The PI should be read on the appropriate channel set to the peak/area and not log scale.

Formalin fixed, paraffin embedded mouse colon stained with Rat anti BrdU antibody

Fig 2. Formalin fixed, paraffin embedded mouse colon stained with Rat anti-BrdU antibody

Immunohistological staining with anti-BrdU antibodies

Bio-Rad’s anti-BrdU Clones Bu20a and BU1/75 (ICR1) can be used for labeling paraffin-embedded tissue sections fixed in formalin. Denaturation of the DNA is critical for successful staining of BrdU. This can be achieved by exposing cells to heat or acid. For heat-induced epitope retrieval, 10 mM citrate buffer pH 6.0 is recommended. Alternatively, 30 minute incubation with 2 M HCl can be performed. The HCl must then be neutralized for 2 minutes with 0.1 M Na2B4O7. Pretreatment of tissues with Proteinase K should be avoided. For clone Bu20a, acetone fixed frozen tissue sections and cell preparations can be used.

Please note ‎that these methods are provided as a guideline, and adjustments may be necessary for your experiments. ‎


BrdU antibody kits for measuring DNA fragmentation during apoptosis

Bio-Rad also offers two kits for measuring the level of fragmentation of DNA during late stage apoptosis. Both kits are called APO-BrdUTM TUNEL assay kits; one is specific for immunohistochemistry, and the other is used for flow cytometry. Read our kit description documents for detailed explanation of contents, components, protocols, FAQs and references.

Guide: Apo-BrdU™ flow cytometry & microscopy kit

Apo-BrdU™ flow cytometry & microscopy kit

Fig 3. Flow diagram of the APO-BrdUTM flow cytometry apoptosis assay

Guide: Apo-BrdU™ - immunohistochemistry (IHC) kit

Apo-BrdU™ - immunohistochemistry (IHC) kit

Fig 4. Flow diagram for the APO-BrdU-Immunohistochemistry (IHC)TM apoptosis assay

Protocol: Apo-BrdU™ flow cytometry and microscopy kit

Protocols within this guide include:

  • Cell fixation protocol
  • Protocol for measuring apoptosis in the positive and negative controls
  • Flow cytometry protocol
  • Staining of tissue cryosections (TCS)
Protocol: Apo-BrdU™ - immunohistochemistry (IHC) kit

Protocols within this guide include:

  • Staining of paraffin embedded tissue (PET)
  • Staining of cell preparations fixed on slides (CFS)
  • Staining of tissue cryosections (TCS)
 

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