[Histonet] Hematoxylin Precipitate

[Gayle Callis]

Sandy, 

After years of using Richard Allan's hematoxylin 2 with great success,   if
we didn't filter daily before use, we had stain precipitate on sections.
Some of this comes from the hematoxylin continuing to oxidize in open air,
bacteria and other "crud".   Tim is absolutely correct ignoring
manufacturers no filtering instructions.   Being old school, we were taught
to faithfully filter any hematoxylin, regardless of progressive or
regressive types.    If we topped off hematoxylin 2 or used new stock,  the
stain was filtered into a CLEAN staining container/dish.  Keep an extra
container around if possible.   We used a medium fast filter paper, Whatman
54.   I realize this takes time but junk on a slide is NOT good thing,
especially after IHC staining and have a photo to show this - the result of
being lazy and not filtering the hematoxylin on that particular day.       

We used a distilled water rinse before hematoxylin2, but DI H2O will be
contaminated with cellular debris and last hydrating alcohol carryover.
Change DI water frequently if you have many runs in a day.   We used 1
minute running tap water rinses after hematoxylin, clearant and bluing.   If
you are using running water rinses, take a look at the blue ppt in the post
hematoxylin container as you don't want that sticking to sections.   Non
running water rinses should be changed after each H&E run in my opinion.
Adequate clean water rinses are important to not have carry over of clearant
into bluing reagents or bluing reagent into eosin in order to maintain
correct pH for staining.    

Good luck

Gayle M. Callis
HTL/HT/MT(ASCP)  

-----Original Message-----
From: Tim H via Histonet [mailto:histonet at lists.utsouthwestern.edu] 
Sent: Tuesday, September 22, 2015 11:25 AM
To: histonet at lists.utsouthwestern.edu
Subject: Re: [Histonet] Hematoxylin Precipitate

You should be filtering your Hematoxylin on a daily basis regardless of what
the manufactures says.  We use to filter twice a day since we did a
traditional overnight run and then again in the afternoon for specimens that
had been microwave processed.  So much tissue washes off in the solutions
they should be changed or filtered fairly regularly to try and prevent cross
contamination on the slides.
 
You can also try increasing your rinse times and see if that doesn't help as
well.  
 
Thanks,
 
Tim
> 
> Message: 1
> Date: Mon, 21 Sep 2015 15:14:39 -0500
> From: "Sandra Cheasty" <cheastys at svm.vetmed.wisc.edu>
> To: "Histonet (histonet at lists.utsouthwestern.edu)"
> 	<histonet at lists.utsouthwestern.edu>
> Subject: [Histonet] Hematoxylin Precipitate
> Message-ID: <4cda87133587e64c965ce6c356d18f59 at svm.vetmed.wisc.edu>
> Content-Type: text/plain; charset="us-ascii"
> 
> Hello all,
> 
>                 Has anyone using Richard Allen Hematoxylin-2 noticed an
odd artifact on the slides after using the Hematoxylin for more than a few
days on their stainer? We are seeing small spore or pollen-like blue dots
here and there on the slides. It is not coming from the water bath or our
water supply on the stainer. I used sterile gloves, opened a new case of
slides, dipped them in DI water, then in the RA Hematoxylin 2 on the
stainer, then in DI again, air-dried and coverslipped them, and the blue
dots were there. The only way we got rid of the blue artifact was to use new
RA Hematoxylin-2 every 2-3 days, which is a bit expensive.
> 
>                 Thanks for your input, and if you can recommend a
different, reasonably priced hematoxylin, that would be awesome.
> 
> Cheers,
> 
> Sandy
> 
>  
> 
> Sandra J. Cheasty, HT (ASCP)
> 
> Histology & Necropsy Supervisor
> 
> UW-Madison, School of Veterinary Medicine

 		 	   		  
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