after PFA fixation, EGFP intensity fated away over time - Anyone noticed this? (Dec/14/2007 )

Hi, guys:

I had some cells transfected with EGFP vector, checked ok after 24 hour with a lot of cells positive. I fixed them with 4% PFA and was going to take some pictures later on. To my surprise, after a three-day storage at 4 C in the dark, the intensity of EGFP decreased dramatically. Anyone had the same experience in the past? Thanks.

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-genehunter-1-

Whats the pH of PFA solution? I know if its not 7.4 or close, there could be loss of GFP fluorescence.

-scolix-

Yeah, Its little off. Way off, I meant.

Thanks for the hint, scolix, as always.

You would not have any prob with PFA at pH7.0 ?

-genehunter-1-

QUOTE (genehunter-1 @ Dec 14 2007, 04:36 PM)

You would not have any prob with PFA at pH7.0 ?

i havn't really tried it out at 7.0, I always make it up to pH 7.4.

-scolix-

I don't know for GFP but I have the same problem with FITC when the solution is slightly acidic.
I would stay close to pH7.4 rather than 7.0

-Missele-

I've had cells with EGFP stored for more than a month in PBS + 2%PFA (pH was 7.4) and no loss of intensity noticed. Why do you actually need 4% PFA? There are severall fluorescent proteins that are loosing fluorescence intensity upon fixation (e.g. for DsRed this effect is quite dramatic), so using less concentrated PFA might help?

-vairus-

If I would have to make a bet, I would say that the problem is more due to acidic pH than PFA. However I don't know the truth.

-Missele-

QUOTE (genehunter-1 @ Dec 14 2007, 01:27 PM)

do you use DABCO or other reduction solution?

-The Bearer-

QUOTE (The Bearer @ Dec 16 2007, 03:55 AM)

QUOTE (genehunter-1 @ Dec 14 2007, 01:27 PM)

do you use DABCO or other reduction solution?

No, I did not.

I think I did not adjust the pH to neutral. EGFP was either denatured or over corss-linked.

-genehunter-1-

The problem is pH. We always check the pH of PFA solution and adjust up by NaOH if necessary. Keep about 7.3 - 7.5

-Almasy-