Re: Poly - L -vs- Amino Alco (++ Slides)
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|From:||"J. A. Kiernan" <firstname.lastname@example.org> (by way of Marvin Hanna)|
On Mon, 17 Jan 2000, Dan Diaz wrote:
> Does anyone out there know the EXACT differences between Poly-L Lysene
> Slides and Amino Alco Salene(SP?)A.K.A Positively Charged or Plus Slides?
Polylysine (it doesn't have to be poly-L-lysine) is a synthetic
peptide: a polymer with multiple amino groups, many of which are
protonated (positively charged) over quite a wide range of pH.
This makes them stick to glass, which has a surface covered in
negatively charged silicate ions (except when immersed in a
medium of low pH - acidity discourages the ionization of weak
acids - but this doesn't seem to matter much). There are plenty
of free amino groups, belonging to parts of the polylysine
molecule that are not in contact with the glass.
A slide smeared with polylysine is therefore covered with
positively charged sites, and these will attract negatively
charged sites (anionic groups) in sections or on the
surfaces of whole cells. Suitable negatively charged groups
are carboxyl groups of proteins and carbohydrates (but only
in neutral or alkaline media), phosphates of nucleic acids
(same remark applies) and half-sulphate esters of connective
tissue matrix and many secretory products (negatively charged
at any pH because H2SO4 is a strong acid). A tissue rich in
strongly acidic groups, such as cartilage, derives the most
efficient adhesion, especially in acid conditions, where
the number of protonated amino groups is greatest. Acidity
suppresses attraction between polylysine and glass, but this
doesn't seem to matter too much in practice.
Aminoalkyloxysilanes (I think that must be what you mean) provide
a positively charged glass surface by a different mechanism.
The usual reagent (and the only published one, to my knowledge)
is 3-aminopropyltriethoxysilane, also known as APES. Clean slides
are dipped in a 2% dilution of this liquid in acetone (this
contains traces of water, which is essential for the reaction). The
ethoxy-silicon part of the APES molecule reacts with water and the
rather unstable product of the reaction combines with the silicic
acid groups of the glass surface, releasing water as a by-product.
This produces a glass surface with amino groups (from APES)
joined by covalent bonds (very strong) to the silicon atoms of
the glass. Unlike the ionic attraction between polylysine and glass,
the attachment of APES is strong even at low pH.
> IF so. What are the useful applications of each and why are they used in
> these applications?
Both are used as adhesives, to reduce the chances of sections
(or cells etc) coming off slides during staining procedures.
As you will gather from the foregoing remarks, adhesion is
generally stronger in acidic than in alkaline conditions,
because alkalinity suppresses the protonation of amino groups.
> I also need to know why is one used when the other
> cannot be used.
They both work the same way, so it's unlikely that there are
absolute exclusions for either. APES should provide better
adhesion than polylysine in a modern Feulgen hydrolysis (using
5N HCl), and you might expect polylysine to be degraded in
methods that involve treatment of slides with proteolytic
enzymes. These are theoretical predictions. Perhaps someone
reading this has done a controlled comparison. If so, let us
> The answer has eluded me for way too long ...
Answers in matters like this always include conjecture, anecdotal
evidence and claims of superiority (based on anecdote and
conjecture) by companies selling rival products. The subject of
section adhesion is discussed critically in various textbooks of
histological technique and microtechnique. Review articles, and
original papers appear from time to time in magazines and in
peer-reviewed journals. If you want to do some reading, let me
know and I'll send you a list.
Most or all of us who do histochemical tests involving extremes
of pH, high temperatures or effervescent reagents would probably
agree that an alkaline solution is much more likely to remove the
section than an acid one. I've never used polylysine, but can
state with feeling that APES isn't much good for jobs like an
arginine blockade with benzil or for staining with biebrich
scarlet at pH>10. Chrome-gelatin is a better adhesive when the
enemy is alkaline. This is of course just another anecdote. It
does make sense (to me) in the field of chemical conjecture,
but that's another story and it's not an answer to Dan Diaz's
question about polylysine and/or APES.
For what it's worth, IMHO and 100% anecdotal: Home-made
chrome-gelatin is better than APES or bought silanized
slides, and _much_ better than Mayer's albumen.
John A. Kiernan,
Department of Anatomy & Cell Biology,
The University of Western Ontario,
LONDON, Canada N6A 5C1
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