As PFA fixation is known to denature proteins and GFP fluorescence is highly dependent on its structure, so would PFA fixation will hinder my imaging experiment where I have a GFP tagged protein expressed in a cell line?
Hi @Puneeta, I would suggest using 1% PFA if you are planning on doing IHC. I am guessing here 4% PFA will quench the fluorescence or maybe hinder the GFP rapidly.
Hi @Puneeta, I would suggest using 1% PFA if you are planning on doing IHC. I am guessing here 4% PFA will quench the fluorescence or maybe hinder the GFP rapidly.
Another option is to use a GFP antibody (alongside proper secondary controls) to amplify the signal. Good luck!
The cross linking that occurs with PFA is unspecific and can denature your GFP, thus resulting in no fluorescence. Try lowering your PFA or look for non-denaturing alternatives.
With transgenic zebrafish embryos I have experienced that the GFP fluorescence only stays specific if the 4% PFA fixation time did not exceed 1 h. Otherwise the whole fish is green. I cannot tell if it´s the same with cells.
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