Re: PAS problems (keeping times, washing)

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From:"J. A. Kiernan" <>
To:Gayle Callis <>
Date:Wed, 07 Apr 1999 01:30:20 -0400 (EDT)
Content-Type:TEXT/PLAIN; charset=US-ASCII

On Tue, 6 Apr 1999, Gayle Callis wrote:

> Also, Culling taught me to make up the periodic acid fresh, daily!
> It is not stable forever.  

    As one who does PAS at long intervals and on a small budget,
    I must own up to keeping my 1% periodic acid solutions for
    as long as possible, and in a clear glass bottle, and using
    it more than once. I do change it if it acquires a colour!

    Just last week I did PAS on some pathological rat CNS sections,
    and they clearly showed the normal capillary basal laminae (quite
    a severe test of CNS PAS) and also the delicate pial connective
    tissue apparently limiting the spread of an inflammatory exudate.
    The periodic acid solution was full-term (9 months old) and had
    been used 3 or 4 times.

> . . .  we never put working Schiffs
> back into the stock bottle (have seen that done!)

    You would see it done every time in my penny-pinching domain,
    unless the Schiff had acquired a pink tint while it was out
    on the bench. If it's not pink and doesn't contain obvious
    bits of debris, why not use it again? Schiff deteriorates
    by (a) losing sulphur dioxide: inevitable if used in open
           vessels. For small-scale work, preventable by using
           it in a screw-cap coplin jar.
       (b) reacting with aldehydes in organic contaminants such
           as bits of sections or residual formaldehyde or
           glutaraldehyde in thick, inadequately washed frozen
           sections. This is likely only to occur in the hands 
           of thick, unwashed graduate students ...
       (c) losing acidity, from collecting hard tap water
           carried over with racks of slides that have been washed 
           but not shaken before dunking them in the Schiff.

> If the Schiffs is turning pink before use, it may also be bad,

    Not may be, must be. Pink Schiff is NBG.

    If you make your own Schiff's reagent, you may well get a pale
    yellow-brown solution. This is aesthetically unsatisfactory but
    functionally OK. (You can remove the brown contaminant by
    shaking with activated charcoal, then filtering.)

> and do not freeze it.

    That's news for me. What happens if you freeze it? Although it's
    customary to keep Schiff in the fridge, this isn't urged by the
    vendors of commercial solutions, and it seems to keep perfectly
    well at 20 C. If freezing does something nasty to Schiff, then I
    think we should be told!  

> Culling also taught us to just wash with running tap instead of
> sulfurous acid rinses, works just fine.

    This is well documented in the refereed literature. The washing
    needs to be very brisk, or there's a risk of nonspecific pink
    background from coloured compounds released in the decomposition
    of diluted and neutralized Schiff's reagent. There also are 
    published claims that bisulphite rinsing reduces the intensity
    of a subsequent Schiff reaction. 

> Gayle Callis

    Gayle:  Are you a pupil of the late Chas Culling? If so, this
    may account for much of the wisdom that you regularly dispense
    on HistoNet. His textbook gave me much inspiration, and I regret
    never having met him in person. His death in middle age gave me
    yet another reason, perhaps the one that made me do it, to give 
    up smoking.

 John A. Kiernan,
 Department of Anatomy & Cell Biology,
 The University of Western Ontario,
 LONDON,  Canada  N6A 5C1

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