BrdU Protocol

Identification of the labeled cells can be applied both in vitro and in vivo to produce a nuclear staining pattern. We provide a method of the 5-bromo-2'-deoxyuridine (BrdU) protocol.

• Tissue and cells are fixed with 4% paraformaldehyde in vitro for 30 mins at 4ºC, in vivo post-fix for two hours at room temperature (r.t.).
• Following fixation, wash in 0.1 M phosphate-buffered saline (PBS) (pH 7.4) with 1% Triton×100 (3 x 5 mins).
• Incubate in HCl (1 N) for 10 mins on ice to break open the DNA structure of the labeled cells.
• This is followed by HCl (2 N) for 10 mins at r.t before moving them to an incubator for 20 mins at 37°C.
• Immediately after the acid washes, Borate buffer (0.1 M) is added to buffer the cells for 12 mins at r.t.
• Samples are then washed in the wash in 0.1 M PBS (pH 7.4) with 1% Triton×100 (3 x 5 mins) at r.t.
• Incubate in 0.1 M PBS (pH 7.4) 1% Triton×100 Glycine (1 M) 5% normal goat serum (1 hr) before incubating overnight (r.t) with anti-BrdU or a combination of anti-BrdU and other antibodies.
• Following the incubation overnight wash in 0.1 M PBS (pH 7.4) with 1% Triton×100 (3 x 5 min).
• Samples can then be treated with a variety of secondary antibodies to visualize the anti-BrdU labeled cells including HRP-conjugated secondaries with diaminobenzidine (DAB) or fluorescently conjugated antibodies.

Creative Bioarray Relevant Recommendations

• Creative Bioarray offers a variety of reagents for studying cell proliferation, cell viability, and cell cytotoxicity including fluorescent and non-fluorescent reporter dyes.

• The protocol should be optimized based on the specific cell type and its growth conditions. Different cell types may require adjustments in the BrdU labeling duration, concentration, and other experimental parameters.
• The length of time for BrdU incorporation into DNA should be carefully selected to ensure efficient labeling of newly synthesized DNA without impacting cell viability or the cell cycle.
• Proper fixation and permeabilization of cells are essential for preserving the cellular structure and enabling the access of antibodies to BrdU-labeled DNA.

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