[Histonet] Re: annoying crystals on sections

David A. Wright dw18 <@t> uchicago.edu
Tue Aug 21 16:27:22 CDT 2012

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 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 Johnson" <ewj <@t> pigsqq.org>
>Subject: [Histonet] annoying crystals on sections
>We are having problems with crystals precipitated on our slides which are H&E 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

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