GFP Additionall info

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From:Jamie Erickson <>
Date:Wed, 28 Apr 1999 17:28:47 -0400

Hi All, (this is a long one)

          Here is what I known about the GFP that our molecular group used in our GFP experiments, the GFP is from Clonetech (pEGFP-n1 MCS cat # 6085-1). I also use Ethanol and Xylene in the VIP for the processing.  
       We have used GFP in almost every tissue except bone, all paraffin sections. Sections are de-paraffinized and dehydrated to water and mounted with Vectasheild with DAPI.  Note, the vectasheild mount does not harden like aqua mount so you have to be careful touching the coverslips.
GFP localization was evaluated on a Nikon E800 Eclipse upright fluorescence microscope, using the FITC filter set.  Images were acquired digitally with a SPOT low-light cooled CCD camera.  Beta-gal expression was observed microscopically, using a light microscope.
         The first GFP experiment we did that worked with paraffin processing was one to evaluate GFP as an alternate method of in-vivo detection of transfection. We used B-gal in one mouse and GFP in another to see if we could get comparable expression. 
We only looked at day 4 but previous results with B-gall showed maximum liver expression at around day 7. It was hard to compare B-gal and GFP samples, GFP seemed to be specific where the 
B-gal livers were stained very blue and it was hard to tell specifically how many cells or what cells were staining. We really should do a longer time course to find out maximal expression.
I have not tried Immuno for GFP on paraffin samples for GFP due to the success of the paraffin processing
               We also did frozen sections that were fixed for 24 hours in 10% Neutral buffered Formalin and that improved the morphology greatly but this was still not as good as paraffin.  The Journal that this image is the cover of is the  Journal of Toxicologic Pathology   Vol 27, Number 1, Jan-Feb 1999. It is just the cover no article or method of GFP staining. Someone asked about length of time GFP is visualize. I have looked at these livers weeks after and have seen GFP and still a strong signal. I'll try to find the slides and look at them again and see if it is still staining. Will let you know. 



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