Hi,
Here is my question :
I'll have to do confocal microscopy on suspension cells (lymphomas), to see internalization of a membrane receptor.
I have to immobilize them before (I think I will use a poly-lysine attachment protocol, anyone as a good protocol ? )
If they stay alive, I will try to do fluorescent live-cell imaging to see the internalization.
But if they die, I will fix my cells. And I would like to fix them at different defined times after immunostaining to see the internalization.
Anyone has an idea to process ?
Thanks a lot !
hi
I think this will help you.
https://worldwide.promega.com/resources/webinars/worldwide/archive/antibody-internalizationassay/
good luck
Thanks, I will check. It loks interessant.
But I still have a ly fluorescent dye, directly targeting my receptor and which is fluorescent when excited. But maybe I can use both .
It seems you will need to do some trial and error type experiments to determine your exact protocol relative to times for each step. If you use a formaldehyde type fixative you should be able to expose your cells to the fluorochrome conjugated ligand, rinse them in PBS and fix them. After a rinse you should be able to detect fluorescence within the cell.
I think you should fix and permeate them at different defined times. but if you do so. one questions you should be noticed : cells will be more likely aggregation by improper fixed methods and lots of them will be lost.
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