[Histonet] fluorescent proteins (RFP and GFP) native fluorescence

[Gayle Callis]

  Caroline,

The first thing you need to do is go to Clontech website and access the 
Living Colours Manual for both GFP and DsRed.  You can also talk to their 
technical service people when you have problems - they are excellent.

We use a fusion protein with eGFP or  DsRed is from Coral but the latter 
suffers from the same loss of fluorescence when fixed.  We have found GFP, 
eGFP doesn't work well with fixation in general.

I have not experienced either eGFP or DsRed photobleaching or failing to 
fluorescence (I am not sure what you mean by dispersion?)  as long as I 
avoid using acetone or acetone/alcohol based fixatives.  GFP, if you will, 
is as stated on title of that manual, best with living cells, although a 
fresh frozen sections, followed by a very short time in 1 -2 % 
paraformaldehyde often retains its fluorescence.

By sliding microtome, do you mean a vibrating mictrome to cut into fresh 
tissue which can either be left unfixed or perfusion fixation of 
animal?  Free floating sections should work just as well as a frozen 
section mounted on a slide.

We now prefer to cryosection at 5 um (could go thicker if needed), mount 
UNFIXED sections on Erie Gold Plus Charge slides(undecalcified bone is 
sectioned with Cryojane tape transfer).  An unfixed frozen section is dried 
at RT for a short time in dark.  After air drying, the unfixed frozen 
sections are rinsed very gently with Dulbeccos PBS to get rid of OCT,  then 
mounted with Prolong Gold Antifade Mounting Media, ready to use from 
Molecular Probes with DAPI - this is a hard set media.  We seal the edges 
of coverslip with diluted permanent mounting media and NOT fingernail 
polish.  Fingernail polish contains isopropyl alcohol that leaches into the 
PBS, causing the GFP to fade.  Diluted mounting media contains either 
xylene or toluene, NOT miscible with aqueous PBS.  You can purchase Prolong 
Gold with or without DAPI.  DAPI gives us two color fluorescence with the 
DsRed fused protein where we need to see it on that on epithelial cell 
surfaces and blue nuclei with DAPI - a tidy way to get some sense of 
morphology.

Unfixed tissues will have a bit of autoflourescence, and probably more with 
aldehyde induced fluorescence.

You did not say if you were doing confocal laser scanning but if you are 
doing so, and use an inverted microscope, there are some delightful glass 
petri dishes with various size and thickness coverslip bottoms designed for 
inverted CLSM.  Large, thick sections are far more easily viewed inverted 
with these dishes, and can be left free floating. We use these for 
vibratome cut thick 100 um sections stained with fluorophore conjugated 
antibodies (red) and allow the autofluorescence to be the 
counterfluorescence in yellow green colors.  This type of viewing should 
work with GFP also.

There is a confocal listserver you need to subscribe to out of Buffalo 
(Online search brought up University of Buffalo Confocal Listserver) which 
will help you out even more as there others doing the same thing you want 
to achieve.  They can probably go into far more detail about GFP and all 
its chimeras, DsRed, and any instrumentation needed to view these 
bioluminescent proteins and specimen preparation.   You can ask questions, 
search their archives, etc - it is valuable resource.

Good luck, I am not sure I answered your questions but I strongly advise 
you to do to the confocal/Buffalo website.



  At 04:59 PM 9/29/2005, you wrote:
>Hey guys,
>
>I was hoping someone here could give me some advice on their
>experience with various fluorescent proteins.  I need a good marker
>for my viral vector.  I have a humanized GFP, RFP and EGFP.  I would
>like to inject the vector into the tail vein to see which tissue
>lights up.  My hGFP variant is not very strong in vitro, but both RFP
>and EGFP work well.  I have heard that GFP quickly disperses when
>mounted on slides.  However, I have seen some papers that use native
>EGFP fluorescence with cryosections and they don't have problems.
>Ideally I would use floating tissue sections of 40 microns as I have
>easy access to a sliding microtome.  I have heard that RFP does not
>work with fixation at all.
>
>I am open to any sort of advice.  Could someone recommend a protocol
>for visualizing native fluorescence with either EGFP or RFP?
>Specifically, whether to fix or not, the thickness of the section,
>floating sections vs. slide mounts, etc.
>
>Also, if there is another fluorescent protein available that you
>could suggest I would like to hear about it.  I have heard of yellow
>and blue fluorescent proteins as well as a monomeric form or RFP.  I
>am also open to using a "universal" fusion protein, for example actin- GFP 
>if it solves the problem of GFP native fluorescence.
>
>Thanks,
>
>Caroline
>
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Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)