RE: [Histonet] Fluorescence backgound

From:Gayle Callis


I don't need to use the Chemicon reagent for my work, but it new product 
being marketed for autofluorescence reduction and something I had not seen 
before  I might try it IF I thought I was having troubles with lipofuscin - 
the reason I brought it up as someone in a previous message was pointing 
out lipofuscin creates autofluorescence problems in brain.  It was only a 
suggestion, not based on me using it - however the info has been filed away 
for future reference.

  My autofluorescence problems are minimal  since we avoid aldehyde 
fixatives.   We prefer all IFA staining on FS fixed with acetone/alcohol 
mixture or 4C acteone.  However, these fixatives will damage DsRed or eGFP 
(we experience loss of fluorescence with these glowing proteins after 
solvent fixation) so the new gig is to leave FS unfixed, air dry for 30 min 
- 1 hr, then coverslip these unfixed, dry FS with Molecular Probes Prolong 
Gold antifade with DAPI, this is hard set, ready to use.  We did not need 
IFA staining, just trying to see where the DsRed-protein was binding 
certain cells, and the DAPI was a bonus - morphology wise.  The results 
were wonderful.   So many ways -------

I just ran across information from Molecular Probes website this 
week,  part of the Q&A from a "Dr. Tech".  If anyone knows about effects of 
solvents on fluorophores, they are probably the experts - maybe their tech 
services know more about the Chemicon Autofluorescence "buster"!  used with 
their fluorescent compounds.

Q: Will the solvents used for fixing, permeabilizing, or deparaffinizing 
samples degrade the fluorescent dyes on my conjugated cells, antibodies, 
proteins, etc.? How can I avoid degrading my fluorescent labels?

A: Many chemicals commonly used for fixing, permeabilizing, or 
deparaffinizing samples will not degrade fluorescent dyes conjugated to 
your biomolecules and cells. Formaldehyde, glutaraldehyde, formalin, 
detergents (e.g., Triton X-100, saponin), surfactants (e.g., Tween, Brij), 
alcohols, acetone, and xylene do not promote the decomposition of the dyes. 
Note that xylene will quench many dyes, but once the xylene is removed, the 
dyes can still fluoresce. Covalently attached dyes are stable in the 
presence of all of these reagents.
Fluorescent dyes are degraded by extremes of pH (pH <3 and >9 to 12, 
depending on the dye) and strong oxidizing agents. You may wish to avoid 
heavy-metal fixation and picric acid. Many heavy metals can either quench 
fluorescence or promote dye degradation by redox reactions.

At 04:23 PM 8/18/2005, you wrote:
>Has anyone used this? Have you used this yourself Gayle? It worries me 
>that the instructions say to use it after staining and then subsequently 
>do 70% ethanol rinses!
>Any info provided is most appreciated.
>At 11:37 AM -0600 8/18/05, Gayle Callis wrote:
>>I think the Chemicon autofluorescence removal reagent - whatever they 
>>call it, is to get rid of lipofuscins - their website will have the 
>>information how it works.

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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