Can RFP fluorescence still be kept after paraformaldehyde fixtation? I recently wanted to do a IF-ststainig, and as far as I know, GFP fluorescence can be well preserved after paraformaldehyde fixtation. However, I have no idea about RFP.
Usually, you shouldn't have any problems there with standard fixation protocols. If you lose the signal, try a less harsh fixation protocol. And in the worst case, you can always try an immunostaining against RFP.
Of course FPs remain fluorescent after FA fixation. Formaldehyde doesn't affect the chromophore of the protein.
Aldehyde groups covalently bind on gamma-amino groups, such as Lysin, Asparagin or Glutamin. They preserve secondary and tertiary structure of the proteins. As the FP chromophore oxidation is due to protein folding, it is essential to maintain the secondary structure of the FP to maintain FP fluorescence.
Ethanol or acetone denaturate (precipitate, coagulate) FPs, thus the chromophore is also denatured.
But if you first fix with formaldehyde, glyoxal or glutaraldehyde you can maintain FP fluorescence after ethanol or acetone dehydration.
HI,
for me formalin worked to preserve GFP but the problem starts with permeabilsation of the cell. The detergents abolish my Fluorescence so I could only advice you to use anti RFP-Ab or analyse the samples without permeabilisation
Regards
Sven
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