Posted by
Steffen Dietzel on
URL: http://confocal-microscopy-list.275.s1.nabble.com/GFP-RFP-quenching-tp2422621p2428420.html
Hello List,
concerning the nail polish: Having done
experiments on both sides of the Atlantic, I
found that the brands I used in the US would kill
the GFP-Fluorescence while the brands in Germany
did not. Must be some specific component that is
not included in all fomulas. We had this
discussion on the list before, at least once,
like 10 years ago :-) In the US, I alternatively
used candle wax, cut in stripes, put at the edge
of the coverslip and heated with a glowing paper
clip. Or, a hardening mounting medium (ProLong, MolProbes).
Concerning GFP and formaldehyde: I sometimes let
the fixation sit over night or over the week-end
without problems. This was the freshly-made-from-paraformaldehyde type.
And while we are on nomenclature, a bit more wise
cracking: there is no such thing as a
paraformaldehyde solution. Paraformaldehyde is a
polymer (white powder) that is unsoluble. Heat
transforms it to the gas formaldehyde which can be solved in water.
Cheers
Steffen
At 18:21 04.03.2009, you wrote:
>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/--
---------------------------------------------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
Head of light microscopy
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