Re: Poly - L -vs- Amino Alco (++ Slides)

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From:"J. A. Kiernan" <jkiernan@julian.uwo.ca> (by way of Marvin Hanna)
To:histonet@histosearch.com
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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
    know!

> 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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