Rapid processing

From:"J. A. Kiernan" <jkiernan@uwo.ca>

Some recent (and older) Histonet communications have been
about artifacts in sections of paraffin-embedded tissue that
has been moved from fixative to wax in much less than the
usual time. It is assumed (probably correctly) that short
times in alcohols and xylene result in incomplete removal
of water and/or incomplete removal of alcohol, with the
consequence that melted wax cannot penetrate into those
parts of the specimen that still contain some alcohol or
alcohol-water mixture. 

Xylene does not mix with alcohol that contains more than
about 2% water. There must be a similar maximum concentration
of alcohol in xylene, for miscibility with melted paraffin.
(Does anyone know the percentage?) Melted wax can completely 
infiltrate only the parts of a specimen that are soaked in a
paraffin-miscible solvent such as almost 100% xylene, and 
definitely no water. (There are some tightly bound water
molecules that cannot be removed by any solvents from the
macromolecular framework of a tissue. These do not impede
penetration of an embedding medium such as paraffin,
and can be ignored.) To get good paraffin sections you must
dehydrate completely, and then completely remove the dehydrating 
solvent if it is not miscible with melted wax.

Some dehydrating solvents are miscible with water and wax. If
you use one of these there's no need for a clearing agent such 
as xylene. The easiest ones to use are tetrahydrofuran and
dioxane, but these are aliphatic ethers that can react with
oxygen (very slowly) to form explosive peroxides. There's not
much risk if you keep them in the lab for only a few years, 
but safety officers don't like these solvents. Tertiary butyl
alcohol has no such risks, but it's more expensive and it
annoyingly freezes at about 25C or 80F, and it's usually
necessary to provide some external warmth. It's often used
for plant specimens.

When it comes to rapid processing into paraffin, the fastest
and most economical method is chemical (reactive) dehydration
with catalyzed 2,2-dimethoxypropane (DMP), which takes only 
minutes for tiny specimens (half an hour is adequate for a 
2 mm cube, and 10 X the volume of the specimen is sufficient: 
0.5 ml is more than enough for a 2 mm cube). DMP dehydration 
is a single step, at the end of which the specimen is 
equilibrated with DMP that now contains about 10% of acetone 
and methanol (products of the chemical reaction with water). 
These liquids all mix easily with clearing agents such as xylene. 
A 2 mm cube is cleared adequately in two half-hour changes of 
xylene with occasional agitation.

Chemical dehydration with acid-catalyzed DMP has been in the
literature for nearly 30 years, and it's surprising that it 
isn't much more widely used. Its introduction to histotechnology
came by way of scanning electron microscopy and botanical
microtechnique, For many years it was thought that only
small objects could be chemically dehydrated. This is true
if you're in a hurry, but objects as large as 2 cm cubes of
muscle are completely penetrated and dehydrated overnight,
even without agitation, which speeds everthing up considerably.
I'm quoting from a paper by Kevin Conway and me, in Biotechnic
& Histochemistry 74 (1) 20-28 (1998), in which we showed that 
for pieces of meat of various sizes chemical dehydration was 
less expensive than using ethanol and other commonly used 

As a one-step procedure, chemical dehydration may not be loved
by companies that sell versatile machines to dehydrate specimens 
by passing them through changes of alcohols and xylene that are 
used repeatedly until they are exhausted. A machine schedule
for DMP processing would need only 4 or 5 stations before
the melted wax: (1) Fixative, (2) Water, (3) Catalyzed DMP, 
(4) Xylene, (5 - for large specimens only) 2nd xylene. With
ingenuity it could be made even more economical.

Research workers, especially in poorly funded disciplines,
have been cashing in on chemical dehydration for more than
20 years. It's been a real money-saver for tiny specimens
for all that time,  We now know that it can save money
(at the expense of a few hours of time) even for quite big
lumps of tissue.
John A. Kiernan
Department of Anatomy & Cell Biology
The University of Western Ontario
London,  Canada   N6A 5C1

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