Dear all,
I'm examining the cellular localization of aromatic hydrocarbon receptor ,Ahr protein, of control and treated samples
I was expecting rounded dapi shape and cytosolic localization of Ahr protein in control sample.
But the result was a little bit strange
Who can explain the shape of dapi and the localization of Ahr and Dapi ?!
Any explanation and advises will appreciated..
The spectral overlap of DAPI and green signal is a common issue. Try to collect the channels sequentially. First the green channel and then the DAPI channel.
What could be happening is that the concentration of the DAPI dye you are using might be too high and the site and areas surrounding it gets overlabelled. DAPI has a wide emission profile. so if the light below 450 nm is not filtered out, you will see some DAPI signal in the green channel.
In confocal microscopy this issue is less evident but in regular flourescent microscopes try to reduce the excitation intensity for the DAPI laser/lamp and limit use about 0.05 to 2 ug/mL of DAPI. Hope this helps.
AHR protein is localized in the cytoplasm and nuclei of most cells.
I understand that you used antibodies labeled with DAPI to this protein.
Since the protein is located in the cytoplasm of cells, DAPI binds to it and stains the cytoplasm of the cells. And the shape is not spherical, because you have cells of that shape.
Unbound receptor should be cytosolic. The receptor binds to ligand that has been taken into the cytosol by the cell and translocates to the nucleas where it interacts with the mechanism that produces mRNA. A number of older papers have described this phenomenon. Just from memory I would lookup articles written by A. Poland and J. Knutson of the U.W. Madison (1980s or so). I think their classic review article was 1980 or so.
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