BrdU Antibody | Bu20a

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Detection of BrdU incorporation into pulsed-labelled ACNC12 cells using Mouse anti-BrdU antibody (clone Bu20a, MCA2483)

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  • BrdU Antibody | Bu20a thumbnail image 1
  • Mouse anti BrdU
  • Mouse anti BrdU
  • Mouse anti BrdU:FITC
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    BrdU antibody, clone Bu20a recognizes bromodeoxyuridine (known as BrdU or BrdUrd). This BU20a antibody is suitable for detecting incorporated BrdU in a wide variety of cell types and is suitable for use on tissue sections in double-labeling techniques.
    • Product Type
      Monoclonal Antibody
    • Clone
      Bu20a
    • Isotype
      IgG1
    2 Formats Available
      Product CodeApplicationsDatasheetMSDSPack SizeList PriceQuantity
      MCA2483C, F *, P *datasheet pdfdatasheet pdf0.2 mg
      MCA2483
      MCA2483TC, F *, P *datasheet pdfdatasheet pdf20 µg
      MCA2483T
      MCA2483FAF *datasheet pdfdatasheet pdf50 µg
      MCA2483FA
      Summary
      Secondary Antibodies
      Negative Isotype Controls
      Useful Reagents
      Positive Controls
      Histology Controls
      More Images
      References
      Reviews
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      • Mouse anti BrdU antibody, clone Bu20a recognizes the thymidine analogue bromodoxyuridine (BrdU), which can be incorporated into DNA during S-phase of the cell cycle. The BU20a antibody is suitable for detecting incorporated BrdU in a wide variety of cell types and is suitable for use on tissue sections in double-labeling techniques.
      • Intended Use
      • Target Species
        Chemical
      • Product Form
        Purified IgG - liquid
        Purified IgG - liquid
        Purified IgG conjugated to Fluorescein Isothiocyanate Isomer 1 (FITC) - liquid
      • Reconstitution
      • Preparation
        Purified IgG prepared by affinity chromatography on Protein G from tissue culture supernatant
        Purified IgG prepared by affinity chromatography on Protein G from tissue culture supernatant
        Purified IgG prepared by affinity chromatography on Protein G from tissue culture supernatant
      • Preservative Stabilisers
        0.09%Sodium Azide (NaN3)
        0.09%Sodium Azide (NaN3)
        0.09% Sodium Azide (NaN3)
        1% Bovine Serum Albumin
      • Immunogen
        Bromodeoxyuridine conjugated to BSA.
      • Purity
      • Approx. Protein Concentrations
        IgG concentration 1.0mg/ml
        IgG concentration 1.0mg/ml
        IgG concentration 0.1mg/ml
      • Reagents In The Kit
      • Preparing The Antibody
      • Test Principle
      • Buffer Solution
        Phosphate buffered saline
        Phosphate buffered saline
        Phosphate buffered saline
      • Fusion Partners
        Spleen cells from immunised Balb/c mice were fused with cells of the NS1 myeloma cell line.
      • Storage
        Store at +4oC or at -20oC if preferred.
        Storage in frost-free freezers is not recommended.
        This product should be stored undiluted. Avoid repeated freezing and thawing as this may denature the antibody. Should this product contain a precipitate we recommend microcentrifugation before use.
        Store at +4oC or at -20oC if preferred.
        Storage in frost-free freezers is not recommended.
        This product should be stored undiluted. Avoid repeated freezing and thawing as this may denature the antibody. Should this product contain a precipitate we recommend microcentrifugation before use.
        Store at +4oC or at -20oC if preferred.
        Storage in frost-free freezers is not recommended.
        This product should be stored undiluted. This product is photosensitive and should be protected from light.
        Avoid repeated freezing and thawing as this may denature the antibody. Should this product contain a precipitate we recommend microcentrifugation before use.
      • Shelf Life
        18 months from date of despatch.
        18 months from date of despatch.
        18 months from date of despatch.
      • Acknowledgements
      • Regulatory
        For research purposes only
      • This product has been reported to work in the following applications. This information is derived from testing within our laboratories, peer-reviewed publications or personal communications from the originators. Please refer to references indicated for further information. For general protocol recommendations, please visit the antibody protocols page.

      • Application NameYesNoMin DilutionMax Dilution
        Immunohistology - Frozen
        Flow Cytometry(1)1/251/100
        Immunohistology - Paraffin(2)
        (1)
        See recommended protocol below.
        (2)
        See recommended protocol below.
      • Application NameYesNoMin DilutionMax Dilution
        Immunohistology - Frozen
        Flow Cytometry(1)1/251/100
        Immunohistology - Paraffin(2)
        (1)
        See recommended protocol below.
        (2)
        See recommended protocol below.
      • Application NameYesNoMin DilutionMax Dilution
        Flow Cytometry(1)Neat1/10
        (1)
        See recommended protocol below.

      • Where this antibody has not been tested for use in a particular technique this does not necessarily exclude its use in such procedures. Suggested working dilutions are given as a guide only. It is recommended that the user titrates the antibody for use in their own system using appropriate negative/positive controls.

      • Technical Advice
      • Technical Advice
      • Technical Advice
      • Recommended Protocol
        FLOW CYTOMETRY ANALYSIS

        Prepare the following solutions before proceeding:
        Phosphate buffered saline (PBS)
        2N HCl, 0.5% Triton X-100
        PBS containing 0.05%Tween-20
        PBS containing 1%BSA (PBS/BSA)
        10mg/ml Propidium iodide (PI)
        0.1M Na2B4O7, pH 8.5

        1 Add BrdU to the cell suspension in culture medium to a final concentration of 10 µmol/L and incubate for 30 minutes in a CO2 incubator at 37°C.

        2 Wash cells twice with PBS/BSA, and resuspend in PBS

        3 Add cells slowly into 5ml of 70% ethanol at -20°C, mixing continuously (vortex preferred). Incubate on ice for 30 minutes.

        4 Centrifuge at 500g for 10 minutes, decant supernatant, and resuspend cell pellet.

        5 Add 2ml 2N HCl, 0.5% Triton X-100 and incubate the cells for 30 minutes at room temperature (preferably on a rocking platform).

        6. Centrifuge at 500g for 10 minutes, decant supernatant and resuspend in 3 ml 0.1M Na2B4O7, pH 8.5.

        7. Centrifuge at 500g for 10 minutes, decant supernatant and resuspend the cells in PBS/BSA + 0.05% Tween-20. Adjust cell concentration to 1 x 107/ml.

        8. Aliquot 100ul of cell suspension into required number of 12 x 75mm tubes.

        9. Incubate the cells with the monoclonal anti-BrdU at the recommended dilution for 45 minutes at room temperature or overnight at 4°C.

        10. Add 2 mls PBS/BSA and centrifuge the cells at 1000rpm for 5 minutes.

        11. If a secondary antibody layer is required then decant the wash and incubate the cells with the secondary antibody for 30 minutes at room temperature. If no secondary antibody layer is required then proceed to step 13.

        12. Wash the cells after the secondary antibody layer by repeating step 10.

        13. Decant off the wash and add 1ml PBS containing 10µg/ml PI (Dilute the 10mg/ml solution of PI 1/1000 in a suitable volume of PBS).

        14. Analyse cells by flow cytometry following the manufacturers instructions. The PI should be read on the appropriate channel set to the Peak/Area and not log scale.


        IMMUNOHISTOLOGY

        Formalin-fixed paraffin-embedded tissue sections:

        Clone Bu20a 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, 10mM citrate buffer pH6.0 is recommended. Alternatively, a 30 min incubation in 2M HCl can be performed. The HCl must then be neutralized for 2 min with 0.1 M Na2B4O7. Pretreatment of tissues with proteinase K should be avoided.

        Frozen sections and cell preparations:

        Acetone-fixed, frozen sections and cell preparations are suitable for staining with clone Bu20a.
      • Recommended Protocol
        FLOW CYTOMETRY ANALYSIS

        Prepare the following solutions before proceeding:
        Phosphate buffered saline (PBS)
        2N HCl containing 0.5% Triton X-100
        PBS containing 0.05% Tween-20
        PBS containing 1% BSA (PBS/BSA)
        10mg/ml Propidium iodide (PI)
        0.1M Na2B4O7, pH 8.5

        1. Add BrdU to the cell suspension in culture medium to a final concentration of 10 µmol/L and incubate for 30 minutes in a CO2 incubator at 37°C.

        2. Wash cells twice with PBS/BSA by centrifuging at 500g for 10 minutes, decant supernatant and resuspend in a minimum volume of PBS.

        3. Add cells slowly into 5ml of 70% ethanol at -20°C, mixing continuously (vortex preferred).
        Incubate on ice for 30 minutes.

        4. Centrifuge at 500g for 10 minutes, decant supernatant, and resuspend cell pellet.

        5. Add 2ml of 2N HCl containing 0.5% Triton X-100 and incubate the cells for 30 minutes at room temperature (preferably on a rocking platform).

        6. Centrifuge at 500g for 10 minutes, decant supernatant and resuspend in 3 ml of 0.1M Na2B4O7, pH 8.5 .

        7. Centrifuge at 500g for 10 minutes, decant supernatant and resuspend the cells in PBS/BSA + 0.05% Tween-20. Adjust cell concentration to 1 x 107/ml.

        8. Aliquot 100ul of cell suspension into required number of 12 x 75mm tubes.

        9. Incubate the cells with the BrdU antibody at the recommended dilution for 45 minutes at room temperature or overnight at 4°C.

        10. Add 2 ml of PBS/BSA and centrifuge the cells at 1000rpm for 5 minutes.

        11. If a secondary antibody layer is required then decant the supernatant and incubate the cells with the secondary antibody for 30 minutes at room temperature. If no secondary antibody layer is required then proceed to step 13.

        12. Wash the cells after the secondary antibody layer by repeating step 10.

        13. Decant the supernatant and add 1ml of PBS containing 10µg/ml PI (Dilute the 10mg/ml solution of PI 1/1000 in a suitable volume of PBS).

        14. 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.





        IMMUNOHISTOLOGY

        Formalin-fixed paraffin-embedded tissue sections:

        Clone Bu20a 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, 10mM citrate buffer pH6.0 is recommended. Alternatively, a 30 min incubation in 2M HCl can be performed. The HCl must then be neutralized for 2 min with 0.1 M Na2B4O7. Pretreatment of tissues with proteinase K should be avoided.

        Frozen sections and cell preparations:

        Acetone-fixed, frozen sections and cell preparations are suitable for staining with clone Bu20a.
      • Recommended Protocol
        FLOW CYTOMETRY ANALYSIS

        Prepare the following solutions before proceeding:
        Phosphate buffered saline (PBS)
        2N HCl, 0.5% Triton X-100
        PBS containing 0.05%Tween-20
        PBS containing 1%BSA (PBS/BSA)
        10mg/ml Propidium iodide (PI)
        0.1M Na2B4O7, pH 8.5

        1 Add BrdU to the cell suspension in culture medium to a final concentration of 10 µmol/L and incubate for 30 minutes in a CO2 incubator at 37°C.
        2 Wash cells twice with PBS/BSA, and resuspend in PBS
        3 Add cells slowly into 5ml of 70% ethanol at -20°C, mixing continuously (vortex preferred). Incubate on ice for 30 minutes.
        4 Centrifuge at 500g for 10 minutes, decant supernatant, and resuspend cell pellet.
        5 Add 2ml 2N HCl, 0.5% Triton X-100 and incubate the cells for 30 minutes at room temperature (preferably on a rocking platform).
        6. Centrifuge at 500g for 10 minutes, decant supernatant and resuspend in 3 mL 0.1M Na2B4O7, pH 8.5
        7. Centrifuge at 500g for 10 minutes, decant supernatant and resuspend the cells in PBS/BSA + 0.05% Tween-20. Adjust cell concentration to 1 x 107/ml
        8. Aliquot 100ul of cell suspension into required number of 12 x 75mm tubes.
        9. Incubate the cells with the monoclonal anti-BrdU at the recommended dilution for 30 minutes at room temperature.
        10. Add 2 mls PBS/BSA and centrifuge the cells at 1000rpm for 5 minutes.
        11. If a secondary antibody layer is required then decant the wash and incubate the cells with the secondary antibody for 30 minutes at room temperature. If no secondary antibody layer is required then proceed to step 13.
        12. Wash the cells after the secondary antibody layer by repeating step 10.
        13. Decant off the wash and add 1ml PBS containing 10µg/ml PI (Dilute the 10mg/ml solution of PI 1/1000 in a suitable volume of PBS)
        14. Analyse cells by flow cytometry following the manufacturers instructions. The PI should be read on the appropriate channel set to the Peak/Area and not log scale

        IMMUNOHISTOLOGY

        Paraffin sections:

        Clone Bu20a can be used for labelling paraffin-embedded tissue sections fixed in formalin. Pre-treatment of tissues with heat-induced epitope retrieval using 10mM citrate buffer pH6.0 is recommended. Pre-treatment of tissues with proteinase K should be avoided.

        Frozen sections and cell preparations:

        Acetone-fixed, frozen sections and cell preparations are suitable for staining with clone Bu20a
      • Flow Cytometry
        Use 10ul of the suggested working dilution to label 1x106 cells in 100ul.
      • ELISA
      • ELISA
      • ELISA
      • Immunohistology
      • Immunohistology
      • Immunohistology
      • Histology Positive Control Tissue
      • Histology Positive Control Tissue
      • Histology Positive Control Tissue
      • Immunofluorescence
      • Immunofluorescence
      • Immunofluorescence
      • Western Blotting
      • Western Blotting
      • Western Blotting
      • Instructions For Use
      • Instructions For Use
      • Instructions For Use

      Additional BrdU Antibody Formats

      Formats Clone Applications Sizes available
      BrdU Antibody : Purified Bu20a C, F *, P * 20 µg | 0.2 mg
      BrdU Antibody : FITC Bu20a F * 50 µg
      • Copyright © 2016 Bio-Rad Antibodies (formerly AbD Serotec)

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          Useful Reagents

            Recommended Positive Controls

              Histology Controls

                • Application NameReference Images
                  Immunofluorescence

                References

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                BrdU Staining Protocol

                Recommended guidance for staining with Mouse anti-BrdU antibody for flow cytometry and immunohistochemistry on paraffin-embedded tissue sections fixed in formalin.

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