Animals given 1.5mg BrdU pulse at least 2 hours prior to transcardial perfusion with saline followed by 4% paraformaldehyde. Sections were cryoprotected, frozen and cut at 15µm on cryostat (NOT PARAFFIN EMBEDDED). Amplified GFP in tumour cells with anti-GFP antibodies followed by Alexa488 conjugated secondary antibodies prior to BrdU protocol as HCL destroys GFP fluorescence. Used BrdU protocol as published by abcam with the modification of a single 30min incubation at 37C in HCL rather than at different temperatures. For development of fluorescence we used Perkin-Elmer tyramide signal amplification for biotin followed by Alexafluor546 conjugated streptavidin. This product is extremely clean. Beautiful staining. Positive control was RMS/SVZ.
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Submitted Feb 22 2008
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