Re: adhesives (yr own)

From:"J. A. Kiernan"

On Tue, 11 Sep 2001, Allrenelltech wrote:
> Could you tell me what would be a good adhesive with the use of things that
> would be commonly found in the lab? We currently use gelatin but even with
> the gelatin sections slide off the slides. Could you please advise. Thank
> you and if you send the reply directly to me I would greatly appreciate it.

Vis a vie  Histonet, all'ss well despite some
 conflict among , course! Nobody wants
to miss out on the answers.  We all Delete without 
reading all the emails with obvious spam Subject lines.

If you use gelatin as an adhesive you have to render it
permanently insoluble in water after the sections have
dried onto the slides and before you expose to the
harmful reagent (alkaline solutions, hot water or
whatever). This is done by chemically cross-linking, to
join together the gelatin molecules, which are big long
ones derived from collagen, and make the whole of the
film between sections and slide into one bignormous
molecule. There will also be cross-links (covalent bonds)
between the gelatin film and protein molecules on the 
under-sides of the sections.

You do the cross-linking with formaldehyde in one of
several ways:
  1. Immerse the slides, without removing the paraffin,
     in any formaldehyde-containing liquid, overnight.
     A buffered fixative is OK but the buffer is not
  2. Expose the slides, in an airtight container, to
     formaldehyde vapour. One or two ml of full strength
     formalin in the bottom of a lidded staining tank
     containing a rack of 10-30 slides, and leave 
     for a few days at room temperature. 
       Alternatively do it at a higher temperature
     (an oven at 50-60C) with a pinch of paraformaldehyde
     as the source of formaldehyde gas. If you do it
     this way you need a really airtight container
     such as a really wide screw-cap jar because you
     can't put an oven in the fume hood. Leave for 
     3 hrs (longer is fine) and then open the container 
     in a fume hood.

A less troublesome technique is to make chrome-gelatin,
coat the slides with it and store them dry ("subbed
slides") until you need them. Mount the ribbons from
water and air-dry in the usual way. The chromium ions
cross-link proteins in the gelatin and probably also
form gelatin-to-section bonds. The chemical
mechanism is completely different from that of
cross-linking by formaldehyde, but with both HCHO
and Cr3+ the reactions occur slowly (hours to days).

For more information, got to Pearse's Histochemistry,
Vol. 1 (equally good in the 3rd or 4th edn), or Gabe's
Histological Techniques. There has been much Histonet
correspondence about all this (,
and there's an informal article (with refs) entitled
"Strategies for preventing detachment of sections from
glass slides" in Microscopy Today 99-6: 23-25 (June 1999).
I don't know if there's any way to get back-issues of
that excellent magazine. One of its editors, Phil Oshel,
is a histonetter, so he may well pick up on this and
let us know.

Section adhesion is a significant economic issue, for
obvious reasons.  The fairly simple principles
that determine adhesion or floating off in different
conditions (acid, alkaline, hot, proteases etc) are
not understood by many technicians and most research
workers. Adhesives and specially treated slides are 
not all equal. The adhesion strategy should be matched 
to the anticipated section-removing forces. This is 
often possible if you can think on the scale of big 
molecules and their net electric charges and affinities 
for water or for hydrophobic parts of other macromolecules.

No applied adhesive or modified glass surface can
resist every type of harsh section-lifting treatment.
For each harsh challenge there is an answer, but 
with some challenges (such as performic acid, and
hot alkaline solutions in alcohol-water mixtures)
the best we can hope for is that 1/5 of the sections
will remain on their slides. For more ordinary
procedures there are simple tactics to improve
adhesion. They've mostly been in print for more
than 50 years.
                          John Kiernan.
John A. Kiernan
Department of Anatomy & Cell Biology
The University of Western Ontario
London,  Canada   N6A 5C1

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