[Histonet] does fixation kill native EGFP or RFP
gcallis <@t> montana.edu
Wed Oct 26 10:25:49 CDT 2005
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.
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.
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