Microwave fixation & processing

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From:"Anatech Ltd." <anatech@net-link.net> (by way of histonet)
To:histonet <histonet@magicnet.net>
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To answer some of Pamela Horge's questions about microwave processing of
Zenker-fixed tissue, 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 here.

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.
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 is 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 staining.

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 at room temperature and under MW
stimulation with Preserve, a glyoxal-based fixative (Energy Beam Sciences).
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.

(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.

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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