The cells that we got can produce a GFP signal continuously, but after doing IHC, the strength of the signal become less and not the same as before IHC. I would appreciate it if someone gave me a clue on how to overcome this problem. The first picture is the signal of GFP before IHC and the second one is after IHC.
Hi Mandana, GFP signal can be quenched by fixation. Methanol fixation can completely destroy fluorescence and formaldehyde/formalin has the least effects (although it will still diminish the signal to a degree). I assume your tissue if fixed in the latter? If you are using 10% formalin you might want to try 2% instead.
The brightest signal will be obtained with the shortest fixation time that you can use (formaldehyde generally penetrates tissue at ~ 0.5 cm per hour so 1-2 hours should be sufficient for small tissue sections). Also note that when the cells are fixed they are no longer making GFP - the signal you see is from the GFP made prior to the cells being "killed". This means that the more you look at the tissue under the fluorescent microscope the more the signal will decline (due to photo-bleaching). Therefore use the lowest laser power possible and try not to dwell in any one place too long.
If the signal remains too low to visualise correctly you may have to probe with an anti-GFP antibody and use corresponding fluorescent secondary. Note: over-fixation will also adversely affect antibody binding/signal.
I hope this helps,
Gary
Hi Gary
I appreciate for your answer and useful tips. I completely agree with your comments. I have question about fixation with formaldehyde. I usually use 4% formaldehyde and fixed the tissues overnight , so according to your comment it should be enough to fix the tissue for 2 h instead of 24 h and forward in EtOH and so on. For the latest suggestion to use anti-GFP A.b. We bought it recently but still not test it. I should do it and see how is it going to get stronger signal or not. Thank you so much for your help and informative comments.
Kind regards,
Mandana
Yes if the tissue is 1-2 cm2 the 2 hours should be sufficient (or 4 hours to be sure of fixation). Good luck with your work!
Gary
Thank you so much Gary for your help! I will do and see how is it going. Hopefully, It will work out and give us strong signal.
I had also had good success with short fixation in 4% PFA and using graded ethanol solutions for dehydration. Denatured ethanol has some methanol in it (
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