Thanks all for many useful and helpful suggestions
and interesting anecdotes.
E. Wayne Johnson, DVM
Enruikang AgTech
MOA Feed Industry Centre
China Agricultural University
Beijing
On 8/22/2012 5:27 AM, David A. Wright wrote:
Hi Wayne Histonet
My guess is that Wayne's crystals are Calcium phosphate - the calcium from the
hard tap water, as described, and phosphate from phosphate buffer (sodium or
potassium) in the solution immediately preceding the tapwater rinse. The
variation in crystal deposition would then be in the degree each tissue/
organelle tends to carry over the phosphate into the tapwater wash. Just mix
drops of the solutions together on a slide and see if crystals form.
A brief deionized rinse followed by tapwater should first remove the phosphate
( crystals) and then allow the desired blueing. Alternatively, substitute
TRIS or similar as the buffer in the preceding step and go directly to tapwater.
If you have valuable sections with crystals on them, you should be able to
chelate away the deposits in an EDTA solution, then restain as needed.
all the best!
-David
==
David A. Wright, Ph.D.
University of Chicago Section of Neurosurgery
Original message
Date: Tue, 21 Aug 2012 09:44:26 -0500 (CDT)
Subject: Histonet Digest, Vol 105, Issue 25 Message: 12
Date: Tue, 21 Aug 2012 07:20:06 +0800
From: E. Wayne Johnsone...@pigsqq.org
Subject: [Histonet] annoying crystals on sections
We are having problems with crystals precipitated on our slides which are HE
stains on tissues from pigs. Tissues are fixed in buffered formalin. We had
trouble months ago with formalin pigment and we had resolved that by using ammonia
in EtOH or picric acid in EtOH. Sometimes we receive fixed samples from the field
that are not buffered but presently all of our tissues are fixed in neutral
phosphate buffered formalin.
We moved the Sakura autostainer to a different location under a fume hood on a
different floor of the building to get the solvent odor out of our work area.
Immediately we began to see a tremendous degradation in slide quality due to
what we initially thought was formalin pigment.
We have changed all of the solutions and all of the stains. We find that if we
use Milli-q water instead of tap water for
rinsing (done by hand in that case) we don't see the crystals, but the eosin
staining quality is not acceptable after rinsing in the acidic (ph ~5) Milli-Q
water.
Our tap water is neutral to slightly alkaline and is very hard with calcium.
We do all sorts of tissues for diagnosis of pig diseases. Sometimes the slides
are quite acceptable but sometimes particularly when looking at small
intestine, the crystals are very annoying. The crystals occur randomly on the
slide except that there is a tendency for them to be centered on nuclei
particularly in intestinal epithelium. The crystals are birefringent in
polarized light but seem to be generally clear not dark like the formalin
pigment we had seen before. Neither ammonia nor picric acid remove these, and
now if we use alcoholic ammonia to treat the slides, the slides come out too
blue. Our slides are cut at 4 to 5 microns.
Brain has the least problem, small intestine seems worst.
We have gone back and cut some blocks that previously stained beautifully with
no pigment or precipitate problems
and those slides also now have the same problem, either crystals if washed with
tap water, or poor eosin staining if rinsed with MilliQ water.
Our next step is to examine the slides microscopically at every step and try to
find at which step the problem is occurring.
Any thoughts or similar experiences?
E. Wayne Johnson, DVM
Enruikang AgTech
MOA Feed Industry Centre
Beijing
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