Douglas W. Cromey
email: dcromey@ccit.arizona.edu
In additition to knowing the chemical basis for the autofluorescence, I would also be very glad to hear about the proper post-fixation treatment that reduces aldehyde-induced autofluorescence.
Stefan Gunnarsson
Stefan.Gunnarsson@DEVBIOL.UU.SE
CURING OF UNSPECIFIC (BACKGROUND) STAINING
Blocking of aldehyde groups
Fixation with aldehydes results in the formation of aldehyde groups which
might cause unspecific binding of reagents and
autofluorescens. Below are four bloccking methods briefly presented.
Method Reagent Incubation 1 50 mM NH4Cl (0.134 g /50 ml) in PBS 15 min at RT 2 0.1% NaBH4 in PBS 2 x 5 min at RT 3 0.15 M ethanol amine (485 ml + 50 ml H2O), pH 7.5 30 min on ice 4 100 mM (0.75%) glycine in PBS 5 min at RTTriton X-100
Beside the permeabilizing effect of Triton X-100 it also reduces background staining in concentrations from 0.02% (G. Ocklind, unpublished).
G|ran Ocklind
E-mail: ocklind@bio.embnet.se
G'day,
One possible chemical basis for the aldehyde-induced fluorescence is
that the fluorophore may be the product of aldehydes reacting with
the epsilon-amino groups of tissue proteins, which leads to the
formationof Schiff base compounds. For further info see Hayat, M.A.
1989, Principles and techniques of electron biological applications,
3rd Ed., London, Macmillan Press.
Hope this is of some assistance,
Gerald Little.
Email ANGJL@Medicine.Newcastle.edu.au
Greetings - I am not sure what you mean when you ask for the chemical basis of aldehyde autofluorescence. I can tell you from an experimental point of view that using the middle line of the K-A laser, if your tissue is fixed in a solution containing even as little as 0.5% glutaraldehyde, you will get autofluorescence. However, we routinely use a fixative of 4% paraformaldehyde and in the rhodamine range (middle K-A line), there is no autofluorescence. Hope this helps.
ANDREA ELBERGER
AELBERGER@UTMEM1.UTMEM.EDU