Vinnie Della Speranza wrote:
> something I've wondered about and would appreciate your thoughts.
> what assurance does anyone have that these slides remain charged over time?
> I would think that those amino groups want to attract anything that will neutralize
> their charge and couldn't this happen long before that tissue section ends up on
> the slide?
A good point, and I must say it had never occurred to me
before. On a dry slide, the free amino group is not
positively charged; it is -NH2. In an aqueous environment,
especially an acidic one, a proton is added to make -NH3+.
This favours adhesion of animal tissue which in general has
an excess of anions (-COO- of proteins and sialic acids,
-OSO3- of glycoproteins and proteoglycans, etc) over cations
(-NH3+ of proteins). Non-ionic forces may also come into play,
just as they do between dye molecules and tissue macromolecules,
once the section and the glass are brought into close apposition.
If these thoughts are correct, APES-treated slides should be
stable indefinitely when dry, but if wet they could collect
contaminating anions such as chloride etc. It makes sense to
mount from distilled water, to dilute such anions. The
undisputable enemies of amino groups are aldehyde molecules,
which combine covalently. You shouldn't store your APES
treated slides in the same cupboard as the formaldehyde.
For what it's worth, my experience with home-made APES
treated slides and with Fisher's "Plus" slides has been
that they are good for 18 months. I suspect they'd keep
for much longer, but they get used up. It's difficult to
evaluate section adhesives in the course of regular working,
because most sections will stick to untreated slides. For
vicious alkaline reagents, used in some histochemical blocking
reactions, my impression has been that a thick coating of
chrome gelatin is better than APES or commercial "positively
charged" slides. This is predictable, because at a high pH
amino groups are largely -NH2 not -NH3+ whereas the carboxyls
of tissue proteins are all -COO- and available to form
coordinate bonds with the Cr(III) ions in the chrome-gelatin.
Just a few thoughts. Thanks for stimulating them! John.
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London, Canada N6A 5C1
<< Previous Message | Next Message >>