GFP in bone

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From:Gayle Callis <>
Date:Wed, 09 Jun 1999 09:37:41 -0600

Hopefully, Donna Montague will enter into this discussion along with
Jamie Erickson.  Both have had success with NBF fixation, then dehydration,
clearing and processing into paraffin.  Donna did decalcify her bone, and
had wonderful results.  It will probably depend on the GFP inserted
into the cells, whether or not it withstands and survives exposure to 
alcohol, other solvents.These ladies didn't give me specifics on which GFP was 
used by the molecular biologists, but your GFP supplier may be able to help you
with that.  As pointed out before, GFP is best used for live cell work, but
is crossing into our realm daily. GFP does survive in formalin but for how
long ????? One could do fresh or fixed frozens.  

My GFP does not survive exposure to alcohol, acetone, hematoxylin and
most aq. mounting medias.  I have to rely on frozen sections, usually fixed
with Paraformaldehyde or NBF, then coverslipped with PBS.  Avoid nail
polish (contains isopropyl alcohol, possibly acetone) that can leach INTO
the PBS.  Will be trying a very dilute toluene or xylene based mounting
media to get away from the nail polish solvent problem. Clonetech suggested
using Crystal mount for GFP, which may work for yours, and if doing frozens
with Instrumedics, fills in those "bubbly" polymer gaps seen on the slide
after polymerization occurs.  Worth a try.  Crystal mount caused our GFP to

It also helps to have GFP filters on the UV scope, but
some GFP may have shifts in UV excitation, with FITC filters working
adequately. If you lose the GFP glow, you can try an antiGFP antibody but doing
that on Spurrs might be difficult. Have had mixed reports on AntiGFP success. 

Lets us know if you retain that green glow.

Gayle Callis

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