Hi Gianluca, You don't say how you are unwinding the DNA to get the antibody in. I find that if all you are interested in is BrdU incorporation and DNA content, the best method is to use acid denaturation of the DNA.The technique does have a few pitfalls but once you are over them it is a very reliable technique. The main thing to be aware of it that you need to make sure that all acid is gone because even a small amount left would be sufficient to denature your primary antibody leading to reduction or ablation of BrdU staining. It is also important to titrate both the primary and secondary antibodies - in general you need a higher protein concentration, or lower dilution, for flow than for immunocytochemistry. Also sometimes if there is low incorporation of BrdU a linear amplification of the FITC signal may be appropriate. There are protocols on my website at: http://sci.cancerresearchuk.org/axp/facs/davies/cycle.html Good luck! Derek >Dear all Flowers, >i'm trying to have a BrDU\PI experiment on LOVO cells. >I used a indirect way w\ Fitc coniugated >secondary Ab fot detect Brdu. But i cant' see the signal in Fl1: >only using fl1 in >logaritmic scale i can see the base fluoscence (cause it's the same >than the negative >control) >Can anyone suggest me a good protocol for BrDU\PI stainning? Thanks a lot -- *************************************************************** Derek Davies Voice: (44) 020 7269 3394 FACS Laboratory, FAX: (44) 020 7269 3100 London Research Institute, e_mail: derek.davies@cancer.org.uk Cancer Research UK mobile: 07790 604112 44 Lincolns Inn Fields, London, UK. Web Page: http://sci.cancerresearchuk.org/axp/facs/davies/index.html In tenebris lux ***************************************************************Received on Tue Feb 17 14:18:00 2004
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