[Histonet] annoying crystals on sections

[E. Wayne Johnson]

we are using a Sakura DRS2000 and we are 3 x 3 minutes in xylene and we have
been keeping it fresh.  The staining is good now but we still see the 
crystals.
If it were paraffin we should see unstained spots on the slide I think.

I have gone to an aqueous 1% HCl today after hematoxylin for regression 
and that seems to be cleaning
them up on most of the slides.  I cleaned out some of the plumbing and 
cleaned some calcium out of the pipes.

We are using "Harris hematoxylin" that we purchase.  We have a  tried 
different counterstains but it seems to
make no difference.

We are using a Sakura tissue processor for overnight processing of 
cassettes.  the embedding is going good
and we get nice flat thin sections.  We are fixing tissues with neutral 
phosphate buffered formalin but
still see some formalin pigment.  We are cleaning that up with picric 
acid in etoh.  We find we still need that
and the formalin pigment is brown to dark brown.  These problem crystals 
are round irregular to rhomboidal
some times sort of large and flat about the size of a cell and they are 
clear.  I thought they were formalin pigment
at first and fiddled with the Picric Acid, and tried Ammonia in Alcohol 
to get rid of formalin pigment and finally
decided that it was not formalin pigment.

I thought it might be something from Scott's tap water (Mg++) so i 
dropped that and tried bluing with NH4+
and it didnt help any.  I tried blueing just with tap water.  Nice 
result but still the crystals.

The aqueous HCl seems to be working and is not harming the nuclei so I 
may have a sort of solution
and am calling it calcium crystals in the water until I know better.

I may look for some sort of filter to put in the water line.

E Wayne Johnson DVM
Enruikang Ag Tech
MOA Feed Industry Centre
China Agriculture University
Beijing

On 8/21/2012 7:51 PM, Debra Siena wrote:
> could it be paraffin?
> Debbie Siena HT(ASCP)QIHC
> Technical Manager | StatLab Medical Products
> 407 Interchange St. | McKinney, TX 75071
> Direct: 972-436-1010  x229 | Fax: 972-436-1369
> dsiena <@t> statlab.com | www.statlab.com
>
>
> ----- Original Message -----
> From: ewj <@t> pigsqq.org [mailto:ewj <@t> pigsqq.org]
> Sent: Tuesday, August 21, 2012 05:46 AM
> To: Debra Siena
> Subject: Re: [Histonet] annoying crystals on sections
>
> We just now ran the statlab version protocol from StatLab's website, using an alcoholic eosin.  No doubt that gives a stronger brighter red stain.  However we still see those crystals!
>
> We are suspecting a water problem.  I have been dismantling some of the plumbing and getting some Ca crystals out of the pipes.
>
> We are manually coverslipping.
>
>    
>>   -------Original Message-------
>>   From: Debra Siena<DSiena <@t> statlab.com>
>>   To: 'ewj <@t> pigsqq.org'<ewj <@t> pigsqq.org>
>>   Subject: Re: [Histonet] annoying crystals on sections
>>   Sent: Aug 21 '12 07:46
>>
>>   Is your eosin alcoholic or aqueous?  What is your staining protocol and what reagents are you using?  This information would be most helpful. Also are the sections paraffin embedded and routinely processed in tissue processor?  
>>   Debbie Siena HT(ASCP)QIHC
>>   Technical Manager | StatLab Medical Products
>>   407 Interchange St. | McKinney, TX 75071
>>   Direct: 972-436-1010  x229 | Fax: 972-436-1369
>>   dsiena <@t> statlab.com | www.statlab.com
>>
>>
>>   ----- Original Message -----
>>   From: E. Wayne Johnson [mailto:ewj <@t> pigsqq.org]
>>   Sent: Monday, August 20, 2012 06:20 PM
>>   To: histonet <@t> lists.utsouthwestern.edu<histonet <@t> lists.utsouthwestern.edu>
>>   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 dont 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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>
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