5-BROMO-2┤-DU LABELING/DETECTION KIT II

General description

Cell proliferation may be studied by monitoring the incorporation of a radioisotope, [ ³H]-thymidine, into cellular DNA, followed by autoradiography.
Alternatively, 5-bromo-2′-deoxy-uridine (BrdU) may be used instead of thymidine. Cells that have incorporated BrdU into DNA are easily detected using a monoclonal antibody against BrdU and an enzyme- or fluorochrome-conjugated second antibody.

Immunohistocytochemical assay for the detection of 5-bromo-2′-deoxy-uridine (BrdU) incorporated into cellular DNA.

Specificity

Anti-BrdU monoclonal antibody specifically binds to 5-bromo-2′-deoxy-uridine, and shows cross-reactivity with 5-iodo-2′-deoxy-uridine (10%). Anti-BrdU shows no cross-reactivity with 5-fluoro-2′-deoxy-uridine or any endogenous cellular component, such as thymidine or uridine.

Application

5-Bromo-2′-deoxy-uridine Labeling and Detection Kit II has been used in:

• labeling of tooth roots for histology
• immunostaining of mice frontal sections
• immunofluorescence imaging of hepatocellular carcinoma sections

The kit is used for the detection of BrdU incorporated into cellular DNA by immunohistocytochemistry.

Features and Benefits

• Safe: No radioisotopes are used.
• Easy to perform: Follows a standard immunohistochemistry protocol.
• Sensitive: Denaturation of DNA with nucleases allows for highly sensitive detection of BrdU.
• Flexible: Allows double-labeling protocols.

Packaging

1 kit containing 7 components.

Principle

Samples prelabeled with BrdU are fixed with ethanol, then incubated with a monoclonal antibody to BrdU, which contains an optimized mixture of nucleases. These nucleases generate single-stranded DNA fragments that allow binding of the antibody to BrdU. Next, an alkaline phosphatase (AP)-labeled antibody to mouse immunoglobulin is added, then bound to the anti-BrdU antibody. The sample is then incubated with the AP substrate and NBT/ BCIP, which is metabolized to form a colored reaction product. The sample is evaluated using a phase-contrast microscope.

Preparation Note

Working concentration: Working concentration of the labeling reagent corresponds to the WC of the In Situ Cell Proliferation Kits.
Sample material:
Cell culture: adherent cells, suspension cells, organ or explant cultures. Frozen or paraffin-embedded tissue sections (after in vivo labeling).

Other Notes

For life science research only. Not for use in diagnostic procedures.