RE: [Histonet] Fluorescence backgound
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.
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-4303 (FAX)
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