Hi All,
I need to FACS-sort formaldehyde-fixed nuclei from mouse brain tissue. What is the best strategy for staining of intranuclear protein for immunolabeling for FACS (staining of soluble RFP-NLS fusion protein)? Ideally, nuclei will be fixed after or during homogenization of the tissue. I plan to purify nuclei on sucrose cushion and then permeabilize with Tx100. Intracardiac perfusion with PFA is not an option for me (fixation must start after dissection of the brain).
Comments from people who were successful in similar procedures will be highly appreciated.
Cheers,
Lech
Hi Lech
I found you procedure very interesting and I'm following it to see the answers from more experienced researchers. I was just wondering are you interested in a particular nuclear protein or do you just need a nuclear marker? Is DNA staining a problem for you?
Dear Andre,
I'm interested in enriching for only a subpopulation of nuclei, which express genetically encoded labels (Cre/LoxP-activated). DNA stain is not a problem, as long as the fluorescence doesn't leak into the other channels used for sorting.
Cheers,
Lech
Hi Lech
In that case take a look at these new DNA stains. If your FACS have a UV, a violet or infrared laser it can be of great help.
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