murine tissue processing, interesting thought (fwd)

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From:"LuAnn Anderson" <> (by way of histonet)
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From: Gayle Callis <>
Date: Thu, 18 Nov 1999 15:21:07 -0700
Subject: murine tissue processing, interesting thought

Well said Gayle.  All need to accept that animal tissue is different than
and go from there.

I meant thoughts, and here are a bunch!

I think it is important to remember that animal tissue in general has
little fat content, tends to be very dry whether you use longer or
even the shorter schedules being displayed.

In general, our mouse tissues, at least the size 3 - 4 mm thick, fixed
longer if just for general routine tissue staining, we would reserve the
shorter fixation time for IHC protocols if we needed that.

If you are fixing mouse liver whole, it is advisable to bread slice
the liver into little slices, to insure total fixation, have seen some
livers not very well fixed at 24 hrs, if not the 70% will be finishing
off your fixation and very dessicating.  We prefer a thinner than 4 mm, but
have processed spleen whole, it isn't huge anyway.

We process all animal tissues at ambient temps, to not add to drying
effects, and never store fixed tissues in 70%, just noticed that
mouse embryos fixed then stored this way, processed were showing a
parched looked artefact after cutting.  They cut well, visibly, but
under the scope, yuck! and were not processed by our lab, a tough thing
to deal with.

Average times appros 40 min per station, 70, 80, 95 X 3, 100% x 3,
Clearite 3 (equal to Propar) 1 hour X 2 (to compensate for water clearing
problems) 4 paraffins X 30 min each at 59- 60 C.  Too high a temp can
contribute to dryness, plus too long in paraffin.  Doesn't make
much difference which paraffin is used, they all work pretty well.
Our xylene clearing would be the same as the Propar/Clearite 3, just like
the new subs better.  Do your cassettes have good openings, not the tiny
holed biopsy style, you need good fluid exchange, 21 cassettes does not
overstuff a processor, if you are at capacity and packed too tightly in a
basket, solvent exchange can be impeded.

How hot is your embedding center, are you cooking the tissues in a
cassette storage area, on the warming plate, in hotter embedding
paraffin?  Do you check the temps of your processor paraffins regularly,
Paraplast plus above 62C breaks down, and creates cutting problems, been
there, done that one!  Correct way to test a temp in cassette storage area
is to fill it with paraffin, since the thermometer is probably an immersion

We still face a block, let it sit on a block of ice with water, not
for a long soak, but enough to give some back to the tissue.  Remember that
when a ribbon is cut, as you move it to a water bath, the section will
experience drying as it floats through the air, before it hits the waterbath.
You have to be careful NOT to oversection away what you have soaked, or you
back to the dry tissue, or rub the face of block with ice water on gauze,
a quicker way.

Rodents and worse yet, birds! have very dry tissues.  One tweak was to embed
tissues (mouse) that posses similar cutting qualities, spleen, liver
in same block, and not put intestine or an easier cutting tissue in this
block.  This is for multiple tissue blocks.

Enough of this tirade of the day but maybe some of it helps.

Gayle Callis


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