cross sections of aortic rings are incubated with DAF2 AM (10µM, KREBS HEPES pH7.4 for 1 hour). then aortic rings are washed and challenged with various agonists for 1 hour. the resulting DAF 2 fluorescence is too weak on fluorescence microplate reader. Because i have no fluorescence microscope close to hand, I would like to fix and preserve aortic rings for few weeks until i could use a fluorescence microscope ...is it possible ? how ?
Hello Sylvain, DAF detects NO in live cells. If you fix artery rings before staining then you will obtain no fluorescence emission from DAF. One option is to fix after staining and treatment, but I have no good experiences with such approach. Best results are always obtained with fresh tissue and live cells. Regards, Alberto
Thanks Alberto. I was not clear but my intention is to fix aortic rings after incubation and treatments. i have no flurorescence microscope close to hand. so i would like to measure fluorescence fiew weeks later. Is it possible to do that ?
I think that the results obtained with more than one day in fixed conditions will be unreliable. Look for another approach. There is other probe similar to DAF that emits in orange-red (580-600 nm) that can be fixed and last longer. Is DAR-2, is even more sensitive to NO than DAF. Fixation conditions are PFA 1-2%, time depending on aortic rings size. Is important don´t use higher PFA concentrations because fluorescence can disappear. Hope it helps, Alberto
Thank you Alberto for your clear and very usefull explanation. I will revise my protocol
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