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Chemical/ Small Molecule BrdU conjugated to BSA.
Our Abpromise guarantee covers the use of ab8152 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC-FoFr||Use at an assay dependent concentration.|
|ICC/IF||Use at an assay dependent concentration.|
|Flow Cyt||Use at an assay dependent concentration.
ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.
|IHC-Fr||1/5 - 1/10.
Dilute antibody in 0.15 M phosphate buffered saline with 1% BSA and 1% Na-azide.
|ICC||Use at an assay dependent concentration.|
|IHC-P||1/5 - 1/10. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
Enzymatic antigen retrieval with proteases can also be used.
Cells were pulse labeled with 10 mM BrdU for 30 min, rinsed twice in prewarmed PBS, and chased in prewarmed culture medium, supplemented with 5 mM deoxythymidine. Incorporated BrdU was detected after ethanol fixation of the cells, which were than rinsed once in PBS and resuspended in 2 ml of 0.4 mg/ml pepsin in 0.1 N HCl. After 30 min at room temperature cells were pelleted, resuspended in 2 N HCl, and incubated for another 30 min at 37°C. Cells were rinsed in 0.1 M borate buffer, pH 8.5, and PBS/BSA (1 mg/ml BSA in PBS). Appropriately diluted mouse anti-BrdU antibody (clone IIB5) was added to the cell pellet, resuspended in 100 micro liters PBS/BSA. After incubation for 1 h at room temperature, the cells were rinsed twice in PBS/BSA. For visualization, FITC-conjugated Fab2 fragments of rabbit anti-mouse Ig antibody were added in a 1/10 dilution. After incubation for 45 min at room temperature samples were rinsed twice in PBS/BSA and the cells were finally resuspended in 0.5 ml cold PBS supplemented with 100 microgram/ml RNAse and 20 µg/ml propidium iodide. The samples were allowed to stand for 15 min on ice in the dark before flow cytometric analysis. In the negative control the primary antibody was omitted.
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