How to fix GFP expressing cell to preserve GFP signal in immunofluorescent stain - (Oct/17/2008 )
I would like to fix and section GFP expressing cells. What is the best way to do it in order to maintain GFP fluorescence? Thanks.
What I have seen is fixing with 1:1 Methanol:Acetone quenches GFP but not Paraformaldehyde.
If the pH of Paraformaldehyde is too basic, it quenches as well.
we had problems with methanol fixation.
But using 4% PFA (ph 7.4) for 30min at RT did the trick.
all the Best.
which pH do you recommend for pFA to quench GFP? also pH7.4?
Hope still someone see this post.
I have a new question about GFP: when you do immunofluorescent stain, is anti-GFP antibody needed or we can see it directly under fluorenscent microscopy?
I routinely fix cells with methanol at -20degree for 15 minutes and always maintain good GFP expression. Usually I don't even use an antibody. However, a post-doc in my lab is amazed because in his previous lab the methanol killed the GFP. I'm not sure if it's the quality (we use 99.9% pure) of methanol, the time allowed to fix or the GFP itself (there are now many different mutant forms). I also maintain GFP when fixing with 3.7% paraformaldehyde for 20 mins at room temp. Really, the only time I've had trouble maintaining GFP is when trying to co-stain for BrdU but I've got that one worked out now as well.
I've used a 4% paraformaldehyde fixation for ~30 at RT without too much trouble and was still able to see fluoresence without an antibody.
Both PFA and Methanol are good to preserve GFP signal. When using methanol, fixaction at low temperature is critical. When using PFA, correct pH (7.4) is important.