Re: staining old tissue

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From:"R.Wadley" <> (by way of histonet)
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	Dear John,

	Me & my big mouth!

	The first part is easy, the most common example is amyloid.  Storage of
unstained sections on slides & even tissue in formalin results in the loss
of staining.  "Theory & Proctice of Histological Technques" 3rd Edn.
Bancroft & Stevens, 1990. pp161.  OK to be fair oxidation/reduction are not
mentioned as possibilities, but is is clear that there is some chemical
reaction that is changing the staining activity of the amyloid in stored

	Histology wax is always assumed to be a nice inert storage menium.  In
truth nothing is further from the truth.  Fluctuations in temperature &
humidity over time cause significant changes to the wax.  For a start it
goes yellow!  While modern waxes are better than those used in the past (&
by people trying to be cheap) I don't think any wax is immune to change.
Another point, & I tried this at Uni, is that with perseverence you can
actually get some staining in sections without treating them with xylene or
alcohols.  This damages the total replacement & protection by wax theory

	In relation to immuno, my sources are more anecdotal.  I do know
that as a
basic quality control measure there should be limits on how long unstained
slides could be kept (immuno & histochemical) for staining.  Another point
is that most people who do immuno work prefer freshly collected tissue to
material that has any sort of age attached to it.  Now that can mean that
there is suspicion as to how blocks & slides have been stored.  Antigenic
sites are susceptable to damage by temperature, pH, UV, fixative, time &
humidity.  These factors must cause chemical changes.

	Most changes are likely to be caused by oxidation of molecules on &
in the
tissue.  But under appropriate conditions I cannot see why reduction does
not become an issue.  Not all the air in labs (particularly in big cities)
is as clean as we would like to think it is.  I must admit I am no great
chemist, even if my appreciation of chemical activity has improved over the
years since I left university.

	As to the last point:  N2 is a nice relatively cheap & friendly
inert gas.
 In a laboratory situation I can see that it could be easily used as a
storage medium.  Because it is inert & if it was used to exclude other
gases (ie O2) I can see that it could make an ideal storage medium.



At 10:18 12/08/1999 -0500, you wrote:
>On Wed, 8 Dec 1999, R.Wadley wrote:
>> One of the great problems with precutting & storing tissue is that you
>> expose the tissue to oxidation & reduction reactions with the
>> surrounding environment. ...
>   Rob: Can you provide references or personal experience to support
>   the occurrence of significant atmospheric oxidation affecting
>   stored paraffin sections?  With frozen sections you can expect
>   changes in the lipids that affect some histochemical tests, but
>   which compounds in a paraffin section remain susceptible to
>   oxidation by air?  You also mention reduction, but do not say
>   what reduces what, or how this might affect the binding of
>   dyes or antibodies.
>> ...  If you must store unstained slides & must get good results
>> from them I can only suggest placing them immediately in a container
>> filled with nitrogen gas, temperature of storage - probably cold.
>    Have you tried this?  If you have, you should write up (publish)
>    your results. I will be happy to give further advice.
> John A. Kiernan,
> Department of Anatomy & Cell Biology,
> The University of Western Ontario,
> LONDON,  Canada  N6A 5C1
>   Phone: (519) 661-2111
>   FAX (Department): (519) 661-3936
>   E-mail:

R. Wadley, B.App.Sc. M.L.S, Grad.Dip.Sc.MM
Laboratory Manager
Cellular Analysis Facility
School of Microbiology & Immunology
UNSW, New South Wales, Australia, 2052
Ph (BH) 	+61 (2) 9385 3517
Ph (AH)	+61 (2) 9555 1239
Fax 	+61 (2) 9385 1591

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