Re: slide questions
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From: | "J. A. Kiernan" <jkiernan@julian.uwo.ca> |
To: | "Karen D. Larison" <LARISONK@UONEURO.uoregon.edu> |
Reply-To: | |
Date: | Mon, 19 Jul 1999 11:37:55 -0400 (EDT) |
Content-Type: | TEXT/PLAIN; charset=US-ASCII |
On Fri, 16 Jul 1999, Karen D. Larison wrote:
> Does anyone still use gelatin-chrom alum slides? If so, for what purpose?
Yes. I do, for pretty well everything. I have tried other things,
including positively charged glass surface - bought at great price
and also hone-made with APES. Chrome gelatin seems to be
definitely better than a +vely charged surface for methods that
use quite strongly alkaline solutions (e.g. pH 9 or higher). For
more ordinary purposes there's not much to choose between them,
but this is purely anecdotal - not based on a specially conducted
quantitative investigation (which I hope to do, one day). Slides
that have been subbed with chrome gelatin can be dried and kept
for years in their original cardboard boxes.
Before about 1976 I used Mayer's albumen as an adhesive, following
the only bad advice I was ever given by my otherwise excellent
mentors. At the time of switching to chrome gelatin I did a lot of
silver staining (mostly Holmes' method for axons), with a pH 8.5
overnight incubation at 37C followed by an even more alkaline
development step. With Mayer's albumen or no adhesive 10 to 20% of
sections would be lost; occasionally and inexplicably 50%. With
chrome gelatin it suddenly became unusual to lose even a single
section. (We were doing batches of 60 slides, each with 5 or 6
sections, in each overnight run.)
Since then I've used chrome gelatin subbed slides routinely
for many different staining methods and for immunohistochemistry
(mostly PAP and ABC prodedures, with paraffin & frozen sections
and also whole-mounts nearly 0.5 mm thick). I have not encountered
any method that fails to work because of chrome gelatin. Even
a histochemical technique for chromium in the tissue (introduced
by fixation) works meaningfully.
> I'm wondering if I can abandon this subbing method, and use "plus"
> slides for all applications, or if there is still some utility in
> this subbing method?
If the risk of losing the sections comes from alkaline reagents
(generally much worse than ecids), then the answer is probably
"No." An APES-treated slide (or one coated with polylysine) owes
its positive charge to protonated primary amino groups. With APES
each is bound covalently to the glass by an intervening chain of
carbon, silicon and oxygen atoms. At high pH amino groups are not
protonated and cannot attract tissue anions. [It is possible that
some commercial products have a quaternary nitrogen, which is
positively charged at any pH, but they don't brag about it in
their advertising, so they are probably just selling APES slides
that you could make yourself more cheaply.] The coordinate bond
between chromium and tissue (or gelatin) carboxyl groups gets
stronger with increasing pH. This may also be true of the bonding
of chrome gelatin to glass.
In research work it is usual to collect more sections and
slides than will be used as part of the initial plan, because
you never know what additional examinations might be needed. If
great numbers of paraffin sections are being mounted and archived
for possible staining in the future by unknown methods, chrome
gelatin is probably the adhesive of first choice. It is cheap and
seems to work for nearly every procedure. The only thing against
it is that you have to spend a fair bit of time dunking (or smearing),
drying and re-packing slides.
These operations could be automated, and it is surprising that
chrome gelatin subbed slides are not sold by all the companies
that sell stuff to histology labs. If any such company feels
like marketing pre-subbed slides, I hereby volunteer all sorts of
helpful hints (as a consultant, for a negotiable and hopefully
large fee). Meanwhile, there will be some free advice for the
ordinary workers in the next issue of Microscopy Today. (Possibly
Phil Oshel will follow this up with an enthusiastic invitation to
get all histonetters on the M.T. mailing list.)
Enough said,
John A. Kiernan,
Department of Anatomy & Cell Biology,
The University of Western Ontario,
LONDON, Canada N6A 5C1
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