Microwave fixation & processing

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From:"Anatech Ltd." <anatech@net-link.net> (by way of histonet)
To:histonet <histonet@magicnet.net>
Content-Type:text/plain; charset="us-ascii"

There are many ways to skin a cat, as this forum so often has shown.  I
present the following in the spirit of offering another way, not as
criticism of anyone else's methods or ideas.  Sorry about the delay in
answering Pamela Horge's request for help, but severe weather in west
Michigan knocked out our ISP.

Let me say at the outset that we at ANATECH LTD. have spent many months
researching the entire fixation and processing phenomena under microwave
stimulation, using an Energy Beam Sciences H2800 Processor.  Details of
this research will be available soon, including several color
photomicrographs, and if anyone wishes a copy, please contact me directly.
I will summarize some of the pertinent information below; in a nutshell,
stunningly beautiful and reproducible results are possible.

In our experience, fixation is the all-important factor in MW processing,
just as it is in conventional processing.  Inadequately fixed material just
will not process well, and will usually present difficulties in sectioning.
The second most important factor is tissue thickness.  Without resorting to
vacuum and/or pressure, we have not been able to get consistent, high
quality results on every specimen unless they are grossed no thicker than 3
mm.  Don't guess at this thickness; measure it.  We prefer to use even
thinner specimens, especially for most rapid turnaround time.

Kidney needle biopsies certainly are small enough to avoid the size
restriction, so I must conclude that fixation time is the likely culprit in
Pamela's specimens.  Zenker's is fast, but not fast enough to allow only an
hour or so for fixation, even with needle biopsies.  This is especially
true with zinc Zenker's.  Remember, fixation in the broad sense involves
penetration and chemical reaction.  While a needle biopsy may be completely
penetrated in an hour or two at room temperature, the tissue is certainly
not chemically denatured to the extent necessary for proper processing and

When inadequately fixed tissue is processed, the alcohol used in
dehydration will finish the denaturing process.  This might allow proper
cutting, but nuclear detail and staining characteristics will not resemble
the classic patterns associated with the primary fixative.  If Pamela's
specimens cut OK but lacked nuclear detail or were washed out (any strange
colors?), it is a sure bet that more time in the primary fixative is needed.

The protocol we developed works well with many fixatives (we did not try
zinc Zenker's).  It involves post fixation with Preserve, a glyoxal-based
fixative (Energy Beam Sciences), used both at room temperature and under MW
stimulation.  The schedule for 1 mm thick specimens is as follows (times in
parentheses are for 3 mm specimens):

(1).  Initial fixation in the fixative of your choice, room temperature
(RT); microscopic appearance of the final tissue will depend upon how long
this fixation time is.  If time here is very short, then your specimens
will look as if they had only been exposed to glyoxal.  That is great for
many people, because morphological detail is superb, but if you want things
to look like they had been fixed in something else, you may not be
completely happy.  For extremely fast primary fixation, use Prefer (ANATECH
LTD.); combined sequentially with Preserve, it has produced the fastest
total turnaround times for us.

(2)  Gross into Preserve

(3)  Post fix in Preserve at RT, 20 (30) minutes

(4)  Post fix in Preserve at 55 degrees C for 4 (16) minutes

(5)  Dehydrate in 3 changes of ethanol (Reagent ethanol is fine), 67
degrees C for 4 (8) minutes each

(6)  Infiltrate in 1 bath of wax at 84 degrees C for 7 (28) minutes.

Total scheuduled time excluding initial fixation and grossing is less than
1 hour.

Note that clearing agents are not used.  While wax and alcohol are
essentially immiscible, the alcohol is evaporated out of the specimen and
the wax moves in to take its place.  WITH PROPERLY FIXED TISSUE
(emphasizing, not shouting here), no harm is done to microscopic
morphology, as anyone can attest who has done this successfully,
theoretical experts notwithstanding.

Nor do you need graded alcohols, or even a sequential combination of
ethanol and isopropanol.  Most microwavers do the latter, and it certainly
is fine; it simply is not necessary given the protocaol above.

We use a stacked configuration for our cassettes:  4 cassettes in each of 5
layers.  Total fluid volume is about 600 ml and ramp up time is 1-2
minutes.  In controlled experiments, we were unable to detect any
difference in quality amongst these cassettes, either using 1 mm specimens
on the 1 mm protocol, or a mixture of 1, 2 and 3 mm specimens on the 3 mm
protocol.  Microwaves evidently are penetrating satisfactorily (uniformly)
through the stack of cassettes.  This is not surprising since both
solutions are more transparent (to microwaves) than water.

Perfectly satisfactory and consistent results can be obtained using the
protocols above, omitting Preserve altogether; however, this is possible
only if tissues are COMPLETELY fixed in the primary fixative (even more so
than for conventional processing).  For NBF, allow 12-24 hours for needle
biopsies.  Zinc Zenker's would probably take close to 12 hours (I have not
tested this).  In other words, there is little point in using a microwave
processor without microwave fixation if speed is your goal.

Few fixatives can be microwaved safely.  NEVER microwave formalin
solutions.  You will easily generate localized vapor concentrations at or
above imminently lethal levels.  This is nothing to mess around with, and
cannot be emphasized enough.

One last note on Zenker's and most other fixatives:  their salts are
generally insoluble in anhydrous alcohol, and will precipitate within the
tissue.  This makes cutting difficult.  If you do not use Preserve as a
post fixative, you should rinse specimens well with DI or distilled (not
tap) water.  Tap water may precipitate zinc and phosphate salts.


Richard W. Dapson, Ph.D.
1020 Harts Lake Road
Battle Creek, MI  49015
800-262-8324 or 616-964-6450
Fax 616-964-8084
E-mail anatech@net-link.net

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