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Dear All

Trivia on use of mounting medias with GFP. Unfortunately, our problem with
maintaining GFP fluorescence was another issue and never sure if researcher
used wGFP or eGFP.

Curious about what chemical in nail polish caused GFP to fade, noted one
culprit was isopropyl alcohol which probably leaches into aqueous buffer.
We tried AquaMount, CrystalMount (liquid coverslip on recommendation of
Clontech) and several proprietary aqueous mounting medias on hand.  These
contained Polyvinyl alcohol (MW unknown) and propylene glycol (MW unknown)
but best fluorescent retention results came from using PBS
(Clontech Living Colours Manual) then sealing coverslip with toluene based
permanent mounting media thinned to base coat nail polish consistency with
more toluene. We discarded thicker clear nail polish, washed bottle with
acetone/let dry to keep the tidy little brush for application of thinned
media.  Since toluene is not miscible with water it can't leach into
aqueous buffer to damage GFP or our thoughts on substitute sealant. The joy
was a rapid drying coverslip sealant!

An acquaintance works with eGFP in bone, neutral buffered formalin fixed,
decalcified,and processed into paraffin through normal alcohol gradient and
clearing agent before hot 60C paraffin, and maintained eGFP. Removal of
paraffin involves reversal of processing, so her bone tissue w/ eGFP had
major exposure to alcohols, clearants, and heat without loss of eGFP.

Kusser et al (J Histochem Cytochem, 2003) also decalcified murine bone and
successfully retained GFP.

Good luck with your GFP project


Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
S. 19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

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