GFP/mounting media

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From:Gayle Callis <>
Date:Sat, 03 Apr 1999 17:23:12 -0700

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

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

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