Brdu免疫组化ProtocalProtocol for Brdu staining • 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 0.1M Phosphate Buffered Saline (PBS) (pH 7.4) with 1% TritonX1...
Protocol for Brdu staining • 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 0.1M Phosphate Buffered Saline (PBS) (pH 7.4) with 1% TritonX100 (3x 5 mins) • Incubate in HCl (1N) for 10 mins on ice to break open the DNA structure of the labelled cells • This is followed by HCl (2N) for 10 minus at r.t before moving them to an incubator for 20 mins at 37°C • Immediately after the acid washes, Borate buffer (0.1M) is added to buffer the cells for 12 mins at r.t. • Samples are then washed in wash in 0.1M PBS (pH 7.4) with 1% TritonX100 (3x 5 mins) at r.t. • Incubate in 0.1M PBS (pH 7.4) + 1% TritonX100 + Glycine (1M) + 5% normal goat serum (1hr) prior to incubating overnight (r.t) with anti-BrdU or a combination of anti-BrdU and other antibodies. • Following the incubation overnight wash in 0.1M PBS (pH 7.4) with 1% TritonX100 (3x 5min) • Samples can then be treated with a variety of secondary antibodies to visualise the anti-BrdU labelled cells (Fig 1 and Fig 2) including HRP conjugated secondaries with diaminobenzidine(DAB) or fluorescent conjugated antibodies such as AlexaFlour 488 or 533
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