RE: [Histonet] over vs underprocessing

From:Tony Henwood

My thoughts below

Tony Henwood JP, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager
The Children's Hospital at  Westmead,
Locked Bag 4001, Westmead, 2145, AUSTRALIA.
Tel: 612 9845 3306
Fax: 612 9845 3318

-----Original Message-----
From: louise renton []
Sent: Tuesday, 30 September 2003 18:50
Subject: [Histonet] over vs underprocessing

Dear All,

There have been numerous posts  and replies over time regarding the quality
of processed tissues, with under or over dehydration, fixation or processing
being cited as the cause. I would lke some clarification and correction on
my personal viewpoints on  this issue  as the case may be.

To my understanding:
a) Fixation and dehydration have an absolute end result, ie one cannot
"over" dehydrate, as once the water is gone, it's gone. However, residual
water is immiscible with clearing agents or wax, and would thus produce a
soft poorly processed tissue. Thus "under" dehydration is a real entity.
However, anecdotal evidence has stated that these tissues may become
'alcohol-fixed" thus making them difficult to cut. What is the rationale
behind this?

Alcohol is a fixative as well, so inadequately formalin fixed tissues will fix in alcohols

b) "Over" processing, is the result of excessive or prolonged heat
coagulating proteins rendering the tissue into hard nasty uncuttable little
nodules. I seem to recall this demonstrated in cookery class where eggs were
fried to extinction and thus became an indigestible mass (there are many
proceses and regents used in histology that bear resemblance to cooking

Over processing (a cooked look to tissues) seems to be more pronounced in under-fixed tissue. Tissues fixed in formalin for longer than 24 hours rarely show this change. This is based on microwave processing experience where tissues are regularly under-fixed.

I realise that this might be an oversimplified version of the real
processes, but I am interested in what the experts have to say.

Best regards

Louise Renton
Bone Research Unit
South Africa
Tel & fax +27 11 717 2298
"Time flies like an arrow, fruit flies like a banana"

----Original Message Follows----
From: " Katri Tuomala" <>
To: "Kelly Salceies"
Subject: Re: [Histonet] Paraffin Embedding of Mice Tissue
Date: Mon, 29 Sep 2003 20:03:35 -0400

Hi Kelly,

What is your fixative for these mice tissues and how long? How big (read
thick) are your sections to be processed?
These two things will determine your routine processing protocol.
I'm not familiar with microwave processing, so I won't comment on that.
If the tissue is well fixed, there really isn't a chance to "over process"
it. The problems arise with inadequate fixation, which then leads to
alcohols and xylene drying out the tissue, and hot paraffin then causes
further damage.
Just something to think about....


Katri Tuomala
Anatomic Pathology
St.Joseph's Health Care
Hamilton, Ontaorio, Canada

----- Original Message -----
From: "Kelly Salceies" <>
To: <>
Sent: Monday, September 29, 2003 10:01 AM
Subject: [Histonet] Paraffin Embedding of Mice Tissue

 > Hi Everyone,
 > I am a brand new Tech and am having trouble preping some mouse tissue
 > for paraffin embedding. I have all different tissues (liver, heart,
 > quads, brain, lung, and gastroc) and would like to prep all tissues (if
 > possible)  under the same conditions. I have been using the Shandon
 > Hypercenter XP or the Shandon Histowave (microwave tissue processor...).
 > Does anybody have a good protocol for mouse tissues on either of these
 > instruments?? I have tried a number of protocols, but all my tissue has
 > been really dry in cutting...
 > Any suggestions would be greatly appreciated!!
 > Thanks,
 > Kelly Salceies
 > University of New Mexico
 > Health Sciences Center
 > Dept. of Pathology
 > _______________________________________________
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