Thanks for all of the suggestions. As suggested, we will try to fix the isolated nucleus in ethanol before the 2N HCL treatment. But, if we dehydrate the nucleus first, would the HCL treatment cause more damage to the nuclear membrane since it may take up the acid solution? Just a thought. Also, I have suggested to the researcher to do it like Brdu staining which the cell is intact and fixed, but he really wants to isolate the DNA/nucleus from the whole cell before he stains because he needs to decrease the high bkgd from the cytoplasm as suggested in the "incomplete" protocol he got from a published paper. And with our first two facs trials, it seemed like the 2N HCL damaged also the nucleus/nucleur membrane because I could only detect low signals from FSC (in the same range as debris, except some gave higher signal in SSC.) Thus, I am not sure how to gate only the DNA from the pool of junk when I analyze. Plus, nothing much came out positive for the DNA adduct-FITC staining, only a few percent of what seemed to be background since they fluoresced in all channels. (From the protocol he got, there should be much positive signal and the amount of fluorescence varies depending on different dosages of treatment, which we did not see from the previous two trials. Thus, I think the staining did not work, but we don't know how to perfect it.)
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