GFP in paraffin
|From:||Gayle Callis <firstname.lastname@example.org>|
There was a poster at NSH/Providence by Jamie Erickson et al on processing
tissues containing GFP. Donna Montague has also successfully done this,
but the problem may be the GFP itself, some of the newer chimeras of GFP
are more stable in the presence of solvents, heat and pH which can quench
the GFP fluorescence.
We could not do paraffin, frozens only, and fixed with NBF, simply cut
section, go directly to formalin or PFA, rinse with PBS and mount.
Clontech has a manual called Living Colors that helps you deal with some
We did a mounting media study as our Aquamount and several others also
caused fading of GFP probably due to the PVA and other solvents in the
media. We coverslipped with PBS (a Clontech suggestion) sealed coverslip
with toluene thinned mounting media (or xylene based, thinned with xylene)
as fingernail polish contains isopropyl alcohol that probably leaches into
the aq PBS, toluene and xylene are not miscible with water. I just dumped
out the nail polish, rinsed with acetone, and air dried the fingernail
polish bottle and brush, put thinned mounting media in, loved the little
brush for application!
We also bought a GFP filter for the UV scope, although most people view it
with FITC channel filters.
If you know the GFP is there, you can do IHC with antiGFP antibody, however
that tends to defeat the nifty purpose of glowing GFP, sort of takes the
fun out of it! You can visit with Donna or Jamie on their technics, if you
need them, I have their email addresses.
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717-3610
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