Dear Steve and confocalists,

I tried yesterday paraffin embedding and sectionning of plant
tissues infected with bacteria expressing GFP. I fixed my samples
with 3,7% formaldehyde, and then dehydrated my samples in
increasing acetone concentration baths (30%, 50%, 70%, 100%). Then
I observed them under confocal, and I must say that I did not see
any green fluorescence. Doesn t this problem with GFP observation
after paraffin embedding also comes from the fact that GFP needs
oxygen to fluores? This is a question I asked myself, but was not
able to answer, as some state that oxygen should be only needed
during the maturation steps of GFP. but on the other hand I never was
able to observe GFP after paraffin embedding.
So if anyone would have a trick to do that, I d be as interested as
Steve.
So Steve, I hope this might be of some use for u,
cheers,
tristan
  On  8 Mar 00 at
10:57, Steve Barlow wrote:

> Date:          Wed, 8 Mar 2000 10:57:35 -0800
> Reply-to:      Confocal Microscopy List <[log in to unmask]>
> From:          Steve Barlow <[log in to unmask]>
> Subject:       GFP  and greening tissue
> To:            [log in to unmask]

> hello all
>
> One of my users has GFP labelled protein being expressed in mouse embryos.
> He fixes all his tissue in buffered 4% para-formaldehyde and stores his
> tissue in this soution for months at a time at 4 C.
>
> After a few months, he has noticed some of his tissue, in particular the
> heart, turns green, making examination of tissue for green GFP fluorescence
> impossible.  Has anyone any explanation of this effect?  I have suggested
> he store his tissue in buffered 0.5% formaldehyde for long term storage.
> Any suggestions for a better alternative?
>
> this user also wants to try paraffin embedding rather than continue his
> cryostat sectioning.  Does anyone have a protocol that will preserve GFP
> fluorescence during processing?  ( I thought I saw a thread recently that
> stated alcohol or acetone dehydrations would quench GFP fluorescence.  Does
> this mean any sectioning work  on GFP expressed protein is tissue samples
> relies on a secondary procedure such as anti GFP antibody labelling?)
>
> thanks in advance for your knowledge and insights
>
> steve
>
>
>
>
>
> Dr. Steven Barlow, Associate Director
> EM Facility/Biology Department
> San Diego State University
> 5500 Campanile Drive
> San Diego CA 92182-4614
> phone: (619)594-4523
> fax: (619) 594-5676
> email: [log in to unmask]
> website:  http://www.sci.sdsu.edu/emfacility/
>
> Chairman, Educational Outreach subcommittee
>   promoting access to microscopes
> Microscopy Society of America http://www.msa.microscopy.com/
>

************************************************
Tristan Boureau
Biocenter Helsinki, Department of Biosciences
Division of General Microbiology, PO Box 56
FIN-00014 University of Helsinki, FINLAND
E-mail [log in to unmask]
tel: (00 358) (09)708 59 655
     (00 358) (0)50 329 58 07
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