Non disponible en dehors du Royaume-Uni et de l'Irlande

Application

Samples prelabeled with BrdU are fixed with ethanol, then incubated with a monoclonal antibody to BrdU. The antibody to BrdU supplied with the kit contains an optimized mixture of nucleases. These nucleases generate single-stranded DNA fragments, which allow binding of the antibody to BrdU without destruction of the cellular morphology. A fluorescein-labeled antibody to mouse immunoglobulin is added, then bound to the anti-BrdU antibody. Subsequently, the sample is evaluated using an immunofluorescence microscope.Safe: No radioisotopes are usedEasy to perform: Follows a standard immunofluorescence protocolSensitive: Denaturation of DNA with nucleases allows for highly sensitive detection of BrdUFlexible: Allows double-labeling protocolsBrdU Labeling and Detection Kit has been used for the detection of 5-bromo-2′-deoxy-uridine (BrdU) incorporated into cellular DNA.

General description

The kit is used for the detection of BrdU incorporated into cellular DNA using immunofluorescence microscopy. It is used for the detection of DNA synthesis by either in vitro labeling of cells or organ cultures, or by in vivo labeling, in which frozen or paraffin-embedded tissue sections must be prepared prior to fixation.Normally, binding of the antibody is only achieved by denaturation of the DNA. This is usually obtained by exposing the cells to acid, base, or heat. These procedures result in destruction of cell integrity, including cell morphology and surface and cytoplasmatic markers.The BrdU Labeling and Detection Kit I avoids these problems. The antibody preparation contains specific nucleases which allows access to BrdU after fixation in acidic ethanol. Therefore also simultaneous detection of other markers (double staining) is possible.

Other Notes

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

Packaging

1 kit containing 5 components.

Preparation Note

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.Working solution: BrdU labeling mediumDilute BrdU labeling reagent 1:1000 with sterile cell culture medium (final concentration 10µM).Note: For in vivo labeling undiluted BrdU labeling reagent (1 to 2ml/100 g body weight) is needed.Prepare shortly before use.Anti-BrdU working solutionDilute anti-BrdU solution 1:10 with Incubation buffer.Prepare shortly before use.Anti-mouse-Ig-fluorescein stock solutionDissolve anti-mouse-Ig-fluorescein solution in 1ml double-dist. water.Anti-mouse-Ig-fluorescein working solutionDilute anti-mouse Ig-fluorescein stock solution 1:10 with PBS. If an extended storage is desired, add BSA (bovine serum albumin), 10 mg/ml.Prepare shortly before use.Washing bufferDilute Washing buffer concentrate (10x) (bottle 2) 1:10 with double-dist. water.Storage conditions (working solution): BrdU labeling mediumStore undiluted (1000x) medium in aliquots at -15 to -25°C.Anti-BrdU working solutionStore undiluted antibody at -15 to -25°C.Anti-mouse-Ig-fluorescein stock solutionStable at 2 to 8°CWashing bufferStable at 2 to 8°CSample material: Cell culture: adherent cells, suspension cells, organ, or explant cultures. Tissue sections (after in vivo labeling with BrdU).

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.