I am working with AAV transduced cardiomyocytes expressing an ER standing GFP.
When I fix the cardiomyocytes in 4%PFA and stain them with additional antibodies, the previously strong GFP signal is barely detectable in confocal microscopy.
However, it cannot be due to the fixation with 4%PFA, since the GFP signal is still detectable afterwards.
Only when I permeabilize the cardiomyocytes with 0.2% saponin/tritonx and then stain with e.g. DAPI, the GFP signal almost disappears.
Attached are two images after PFA fixation, one without staining and one stained with DAPI.
As I am a MD student and therefore relatively inexperienced, I would appreciate your help.
Best regards Willem Borbein
I would assume you are imaging at same exposure in both cases. if you are not staining your cells with nuclear protein antibody, you do not really need to permeabilize with saponin. DAPI would stain even without permeabilization. DAPI is very strong nuclear dye. May be your permeabilization is too strong, and its making too big whole on the plasma membrane of your cells. Another thing is, during all of this staining period including the wash steps, you have to avoid exposing your cells to light, you can work under regular sunlight in the lab, but turn off the lamp lights. And store your slides in the dark until analyzing with microscope.
Thank you for your answer Enkhtuya Radnaa !
Yes the exposure time was the same and I worked in a darkened environment.
But if I use other antibodies, such as calnexin-antibodies (for the ER), then I have to permeabilize with saponin right?
I will try it with a lower saponin concentration / incubation time.
in case you need to permeabilize for your experimental set up then it might help to use an additional anti-GFP antibody to amplify the GFP signal. You just need to make sure that the host species for raising the calnexin and GFP antibody are not the same.
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