Re: slide questions

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From:"J. A. Kiernan" <>
To:"Karen D. Larison" <>
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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