Re: tissue processing (times; also clearing agents)

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From:"J. A. Kiernan" <> (by way of histonet)
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On Wed, 17 Nov 1999, Philopena, Jennifer wrote:

> tissues (liver, lung, spleen, kidney, brain, heart).  The livers and spleens
> that I process are consistently very dry.  This is my procedure: 24 hours in
> 10% neutral buffered formalin, store in 70% reagent alcohol, 20min in 80%
> reagent alcohol, 2X 20min in 95% reagent alcohol, 3X 20min in 100% reagent
> alcohol, 3X 20min in Propar (Xylene substitute), 2X 20min in paraffin
> (Paraplast Plus).  I'd like to fix this problem with the tissues being
> over-dehydrated.

   Just a few thoughts:

   Unless your specimens are pinhead-sized, the times you state
   are far too short, and your problem is almost certainly that
   the tissue isn't completely dehydrated (you can't over-dehydrate)
   and isn't sufficiently permeated by the wax. I would recommend
   at least an hour in each of the fluids; (2 hrs would be even
   better if the smallest dimension of any specimen is as much
   as 5 mm), and 3 one- or two-hour changes of paraffin. If you're
   processing with a machine, set it to run overnight and block
   out the specimens first thing in the morning.

   I've not used the clearing agent you mention. Do you know what
   it is? If it's an aliphatic hydrocarbon (decane or similar) it
   will be even less tolerant than xylene of traces of water in
   the last "100%" alcohol. My favourite clearing agent is
   terpineol (synthetic oil of lilac). This would mix with
   alcohol containing 10% water - not that I'm recommending
   anything less than absolute alcohol (which actually contains
   about 0.5% water unless you take steps to remove the last

   Another advantage of terpineol and of some other
   old-fashioned clearing agents like cedarwood oil is that they
   are conspicuously more viscous than xylene etc. Perhaps for
   this reason they are not completely displaced from the tissue
   even after prolonged infiltration in several changes of wax.
   You can smell it (faint, and quite pleasant) when cutting the
   sections. The cutting itself seems more trouble-free (or should
   I say "less troublesome" than after clearing in xylene, chloroform
   or other "thin" liquids, but this is an anecdotal statement and
   probably of little real value. It is undoubtedly influenced by
   what's in the next paragraph.

   Older books on microtechnique (and even books written in the
   1970s by older authors) emphasized the virtues of viscous clearing
   agents, especially cedarwood oil. They recognized that part of
   their advantage was due to difficulty in obtaining absolute alcohol
   in earlier years (a problem long gone by the 1950s), but also stated
   that these clearing agents were necessary for demonstrating
   mitochondria, the smaller kinds of secretory granule and elements
   of the Golgi apparatus. These things needed the thinnest sections
   you could cut. In paraffin this was (and IMHO still is) somewhere
   between 3 and 4 microns, whatever the dial on the microtome says.

   The above statements were fully supported by experienced
   microtomists in the Departments of Anatomy in Birmingham and
   Cambridge (UK) and London (Canada) from whom I learned a lot in
   the 1960s and early 1970s. These guys worked with a great variety
   of tissues, for teaching and research, and had the time to
   change and adjust their techniques to get the best results.

                                       John Kiernan
                                       London, Canada.

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