RE: GFP again (sigh) -- long reply

From:"Montague, Donna C"

Dear fellow GFP sufferers,

Let me begin with the following qualifier: I can only describe what we have
done and the techniques, supplies and equipment we have used that seem to
work on bone for visualization of EGFP. That said, our procedure is as

Osteoblast progenitor stem cells were transfected with a vector containing
an expression promoter and EGFP (Clontech). The resultant transfected cells
were injected into the tibia of SD rats undergoing distraction osteogenesis.
Briefly, an Ilazirov external fixator was placed on the leg with four
K-wires. The tibia was broken approximately 1 cm distal to the growth plate.
The tibia was distracted 1/4 turn q 12 hours for 3 days. The transfected
osteoblast progenitors were injected into the distraction gap using a
tuberculin syringe. Distraction  progressed 1/4 turn q 12 hours for a total
of 14 days. Animals were sacrificed humanely in accordance with IACUC
guidelines. The fixator was removed. The legs were x-rayed , stripped of
skin and fur and placed immediately into an excess volume of 10 % NBF. After
adequate fixation, the legs were decalcified to calcium oxalate end-point
with 5 % formic acid (aq). Specimens were processed by hand according to the
schedule in  R. A. Skinner et al JOH 20 (3): 267, 1997. Sections of the
specimens were floated onto commercially prepared silanized slides, drained
vertically and annealed to the slide on a two-stage slide warmer set at 52
and 58 oC respectively. Slides were deparaffinized in three 5 minutes
changes of xylenes, coverslipped using permount and examined using an Zeiss
Axiovert Confocal Scanning Laser Microscope (CSLM) excitation wavelength 488
 Photos available.

Good luck,Donna Montague, M.S.
Research Associate
University of Arkansas for Medical Sciences
Center for Orthopaedic Research
Departments of Orthopaedic Surgery and 
Physiology & Biophysics

"From: Johnson, Teri Subject: GFP again (sigh)
	We're trying to visualize GFP in mouse tissues.The 4%
paraformaldehyde fixed, formic acid decalcified,paraffin embedded bone
specimens show "staining" in every cell.  The unfixed, frozen skin specimens
show "staining" only in the hair shafts...Donna Montague has had great
fortune in obtaining good results with eGFP.  I'm wondering if maybe walking
next to her or something might help some of this rub off on me? Thanks for
your help!"

And from Martin Weber: "Hi there, we are facing a problem preserving GFP
(EGFP, DsRed, Clontech) visualization in bone sections. To our knowledge we
have to perform a decalcification. Does anybody has experience with a
protocol leaving GFP visualization intact ? Should we use EDTA, formic acid
? In which concentrations ? Preferably we 
would like to perform cryosections rather than parraffin processing. Does
anybody has experience with a method preparing fresh frozen sections of
undecalcified tissues (Kawamoto T. and Masaharu S. Histochem Cell Biol 2000,
113: 331-339). Any help on this problem would be very appreciated."

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