[Histonet] Re: rfp and gfp

Gayle Callis gcallis <@t> montana.edu
Mon Oct 3 10:06:10 CDT 2005


Thank you for your comments and technics, now residing in my GFP file folder.

Gayle Callis

At 07:02 AM 10/3/2005, you wrote:
>Hi Caroline and Gayle,
>Sorry for writing to you both but I think I can help a little on this 
>subject. I just want to echo some of Gayle's comments which are excellent 
>and add a few things.  I currently use both eGFP and mRFP invivo.  I 
>visualise these markers with conventional fluorescence and confocal 
>microscopy.  I cardiac perfuse my rats with PBS (0.1M) and then perfuse 
>with 4% 0.1M PB formalin.  I post fix for 2 hours with the same fixative 
>and cut 40
>mm sections using a slide microtome.  I can see both eGFP and RFP equally 
>well.  Although I will add that RFP is not as strong as eGFP and this may 
>be due to photobleaching/fixation. I have used both free floating and 
>serially mounted sections and saw no difference in 
>visualisation.  However, if you're planning to immuno with serially 
>mounted sections, I would suggest permeabilising with 50% ethanol and 0.3% 
>triton, initially we only used 50% ethanol and saw poor 
>permeabilsation.  You could also use anti-GFP and anti-RFP to enhance your 
>visualisation, I have found this has enhanced our understanding of 
>neuroanatomy/distribution greatly.
>I hope this has helped.
>Patrick Howorth,
>Dept of Physiology,
>University of Bristol,
>BS8 1TD.
>+44 (0)117 33 17112
>patrick.howorth <@t> bristol.ac.uk

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