Posted by
Carol Heckman on
URL: http://confocal-microscopy-list.275.s1.nabble.com/GFP-RFP-quenching-tp2422621p2424209.html
Hi, Scott-
We have found that limiting the duration of formaldehyde fixation to 10 minutes does conserve the GFP signal. It seems to decline the longer the cells sit in the fixative.
Carol Heckman
Center for Microscopy & Microanalysis
Bowling Green State University
________________________________________
From: Confocal Microscopy List [
[hidden email]] On Behalf Of Kelly Lundsten [
[hidden email]]
Sent: Wednesday, March 04, 2009 10:11 AM
To:
[hidden email]
Subject: Re: GFP/RFP quenching
Hi Scott,
Any denaturant will be a potential problem for the protein. This
includes any MeOH that might be used to stabilize the PFA. Have them
make up fresh PFA the hard way, with heat and time, or buy it with a no
MeOH used guarantee. Also, if they are sealing their coverslips with
nail polish, stop that. Use VALAP (Vaseline:lanolin:paraffin) or
something else inert. The nail polish can seep under the coverslip over
time, denaturing the GFP. This sounds like the more likely culprit in
this instance if there is signal right after coverslipping but that
signal fades quickly over time. Or, use an antifade that cures, one
that doesn't require sealant. I believe Prolong Gold fits that criteria
(cures without dehydration around the edges of the coverslip). If the
problem persists after trying these potential solutions, then the PFA
might be changing the efficiency of the GFP due to its cross-linking
effect. Trying lower percent concentration of the PFA would be your
next step.
Good luck,
Kelly Lundsten
Sr. App Scientist
ART, Inc.
-----Original Message-----
From: Confocal Microscopy List [mailto:
[hidden email]]
On Behalf Of Scott Howell
Sent: Wednesday, March 04, 2009 6:52 AM
To:
[hidden email]
Subject: GFP/RFP quenching
List,
We have had some issues here with a lab where the GFP/RFP brightness in
fixed cells has become extremely variable. Like night and day. Wondering
if it may be related to the pH of their paraformaldehyde fix? Does one
particular pH seem to work best?? Any other ideas? Thanks.
Scott J. Howell, Ph.D.
Manager, Imaging Module
Visual Sciences Research Center
Case Western Reserve University
2085 Adelbert Rd.
Institute of Pathology Room 106
Cleveland, Ohio 44106
216-368-2300
http://www.case.edu/med/vsrc/