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.
The following solutions should be prepared:
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
Fig 2. Formalin fixed, paraffin embedded mouse colon stained with Rat anti-BrdU antibody
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.
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.
Fig 3. Flow diagram of the APO-BrdUTM flow cytometry apoptosis assay
Fig 4. Flow diagram for the APO-BrdU-Immunohistochemistry (IHC)TM apoptosis assay
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