Histonetters and Gayle,

Most antifade reagents quench fluorescence, while preventing further bleaching.  
For example, I found that Vectashield quenches the flourescence of FITC by 
about 50% (I used an image processor and a capillary tube full of reagents to 
make these determinations), but is very good at stabilizing this reduced signal 
over time.  That's the beauty of ProLong -- it really doesn't quench the 
fluorescence of FITC much at all, while still providing good anti-fade 
properties.  This is a product I developed when I worked at Molecular Probes, 
so I'm familiar with it's attributes.  It's hard to predict how it would behave 
with GFP, however.  I presume, the GFP protein directs electrons along a 
certain pathway, much like rhodopsin or the phycobiliproteins.  The medium may 
inhibit conformational changes in the protein required for directed electron 
movement, or the antifade reagent itself may wreak havoc with the process.  In 
general, my experience with anti-fades (and I've had plenty of experience) is 
that they are extremely subject to environmental conditions.  For instance, an 
antifade that works well with one antibody may not work well with another.  The 
assumption is that there is some environmental factor at work.  For instance, 
FITC fades extremely rapidly in some environments and is relatively stable in 
others.  I assume the local redox environment is the the primary factor, as 
fluorescence entails the excitation of electrons.

Karen Larison at University of Oregon


Date:          Sat, 03 Apr 1999 17:23:12 -0700
From:          Gayle Callis <uvsgc@msu.oscs.montana.edu>
Subject:       GFP/mounting media
To:            histonet@pathology.swmed.edu

We had the same problem with GFP and aqueous mounting media.  We ended up
testing several aqueous media to see if fluorescence was reduced. Tested were
PBS, PBS/glycerol 1:1, Aquamount, Gel mount, Crystal Mount (actually 
recommended by Tech services at Clontech - GFP gurus) and Vectashield.

Results were the PBS (coverslip sealed with nail polish) maintained the
flourescence best ( and we use GFP filters on the UV scope).  All others 
showed a reduced fluorescence, very disappointing, especially  when
Clontech said should work! The PBS/glycerol use was on sections of brain
that were 300um thick, so the mounting media may not have penetrated this
thick sections easily and the GFP fluorescence was maintained.  My sections
were 5 um containing bacteria with GFP, then added problem of another 
fluorescent marker on cells (IFA staining).  


Molecular Probes sends out a tips on mounting media brochure with purchase of
their Alexa 488 and 546 fluorochromes, that gives info on which media
work best with the Alexa's, FITC and TRITC.  They include their Prolong,
and antifade product.  Since we wanted to do an immunofluorescent stain
on same tissue containing GFP, this info was important.
We basically did not want to purchase their mounting
media, pricey, particularly if it did not help maintain the GFP and have it
sit around being a shelf ornament.

At this point, we live with the PBS mount, excellent for Alexa 488 and
546, take photos immediately on a computerized system.  Hopefully
they have more info on their mounting media and GFP now, my inquiry did
not get anywhere, since they are more involved with their fluorescent
markers/dyes. If you try the Prolong,, I will be interested in your
results.    

One sidelight, the Strepavidin Alexa conjugates are very nice, we like them
better than TRITC, brighter, and works right into our immunostaining
protocols with biotinylated primaries or secondaries.  High dilutions, better
We have GFP in bacteria and did an immunostain on same section using 
Alexa 546 (TRITC replacement) with super results

The GFP seems sensitive to some of the ingredients in mounting media,
polyvinyl alcohol? glycerol? other? but also could be our particular GFP.


Clontech has an excellent booklet on GFP technics, including some of what
has been discussed, for reducing autofluorescence.  PBS was a suggestion.

Question:  are you trying to coverslip tissue sections or cell cultures?

Gayle Callis
Veterinary Molecular Biology
Montana State University
Bozeman MT