I have been working on astrocyte from primary glial cell culture and I want to observe calcium influx after several treatment. I know once Fura-2 AM in the cell, intrinsic esterases hydrolyze the ester bounds and trap the dye into the cell. Therefore loading astrocytes with Fura-2 AM and fixing it by 4% PFA on the coverglass should have work to see the calcium influx happened just right before the fixation, isn't it? Or is the fixation processes by 4% PFA will destroy the membrane and cause Ca2+ fluorescence to be lost?
Yes FURA works, look at my publications v.g. the 1997 review. Mueller cells and astrocytes have the same protein profile plus some extras for the Mueller cells due to their optical properties.
Vera Maura Fernandes de Lima, thank you for your answer. I have checked your publications and I see mostly you load FURA and use perfusion chamber to perform imaging while cells are still alive but not after fixated by 4% PFA. Or may be I couldn't find the right paper. I'd be appreciated if you can send me the link of one of your papers to help me with my problem.
no I never worked with fixated tissue extrinsic fluorescence. sorry, i misanterstood you problem, cannot help further, thanks for your answer to my coment, Vera Lima
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