Re: [Histonet] does fixation kill native EGFP or RFP fluorescence?

From:Gayle Callis


Are you going frozen sections or are you going on with paraffin 
processing?  By saying you are cutting a fresh section, I presumeyou are 
snap freezing a tissue without any fixation prior to the freezing part. OR 
are you snap freezing the NBF fixed liver?  and do you cryoprotect with 25% 
- 30% sucrose after fixation then snap freeze?

By a lot of signal, do you mean everywhere or specific for cells?  You 
could be seeing autofluorescence -  one of the problems with viewing eGFP 
after NBF or PFA fixation.  NBF or paraformaldehyde will preserve these 
proteins, but we always snap froze fresh tissue, then fixed the sections in 
PFA, 2% for only a few minutes.  We had  much better signal if we simply 
cut a frozen section, let it air dry, rinse very gently with DPBS, and 
coverslip the UNFIXED section with Molecular Probes Prolong Gold antifade, 
ready to use (hard set)  in order to protect the fluorescence.  There is no 
law to say you have to fix a frozen section to see eGFP, and we do this 
with DsRED also.  The joy is you have no aldehyde induced autofluorescence, 
merely the natural autofluorescing components found in liver.   We do look 
at these unfixed sections on day of cryotomy,  it takes a bit of 
coordination but it is another way to see eGFP.

Beware of lipofuscins (hey, did I say that correctly?)  This was discussed 
recently on Histonet and you can go to Archives with keyword search to read 
up on this.

We cannot maintain eGFP with any organic solvent exposure i.e. frozen 
section fixed with acetone or acetone alcohol mixture.  When we used 
paraformaldehyde, we had best signal with shortest fixation time and low 
concentration of PFA,  i.e. 2 min fixation of a frozen section versus a 10 
min fixation of a FS in cold 2% PFA in DPBS but our autofluorescence was a 

One way to prove you are seeing eGFP is do an antiGFP on an adjacent 
section, Rockland Goat antiGFP is superb, and you can do this with 
immunofluorescence or IHC.

I suggest you go to the Clontech website and get the Living Colours Manual 
in PDF form, it has a wealth of information for eGFP, etc. on fixation, 
problems encountered and what will autofluoresce in tissues.

Good luck

At 03:46 PM 10/25/2005, you wrote:
>My boss insists that EGFP or RFP expressing tissue will lose the
>native fluorescence if fixed.  Is this true?  I know it isn't true
>for tissue culture, but he is very insistent that it will occur.
>I have cut the liver into blocks and immersion fixed them in 10%NBF
>overnight at 4 degrees celcius..  If I cut a thin section of fresh
>tissue I can see a lot of signal.  He insists I am wasting my time by
>sectioning the blocks.
>Any advice would be appreciated.
>Histonet mailing list

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)

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