[Histonet] Hematoxylin Precipitate

Wed Sep 23 06:55:59 CDT 2015

Well Tim,  
	In the 31 years I have been in histology, I obviously have done all lab work with my eyes closed, never looking at the equipment or solutions I have been using.  I guess my stint in a high throughput contract lab taught me nothing nor did my 25 + years in big Pharma, notorious for hiring every hack who knows what a microscope slide is.  You suggested that I sit around getting published... I am published, BTW, but that accounts for 0.01% of my experiences in the lab, yes, in the lab processing, embedding, sectioning (quite good at that), cryosectioning, performing and troubleshooting IHC and histochemical stains.
 	Something I may have learned, and maybe you will when you catch up, is not assume you know what someone else's work experiences may have been or are.  Histology occurs in many environments with differing equipment, reagents, processing styles, etc.  Some are better than others.  I guess our inadequate processing methods fall short, leaving far too much tissue on the slide where it belongs, and thus we don't have big tissue globs clogging everything, nor do we see precipitate with the hematoxylin used.  Traditional formulations of hematoxylin, sure, absolutely filter.  For anyone who benefits from filtering, why not continue.  No harm will come.  I was merely trying to advise someone who seemed to have an issue I thought I could help with.  Now I realize all issues should be relegated to Tim, the Charles Churukian of our times.  Look him up, if you're stumped by the reference.  Maybe I'll head for Wacky Tabacky land for a vacation.  It is quite beautiful there...

Phil.

-----Original Message-----
From: Tim H via Histonet [mailto:histonet at lists.utsouthwestern.edu] 
Sent: Tuesday, September 22, 2015 5:04 PM
To: gayle.callis at bresnan.net; Histonet
Subject: Re: [Histonet] Hematoxylin Precipitate

Liz, Phillip and all that are
interested,

I take it you have guys never looked at or had someone else
examine what is at the bottom of the Hematoxylin filter after
you put through a day's work.  There will be tissue particles of
tissue along with other contaminates, I am not saying you are going to see
a complete LEEP sitting at the bottom but you will have
contamination.  

Liz, maybe the tissue super adheres up there in wacky tabacky country
and Phillip sitting in a research facility (are you even
involved with processing and staining of slides or just publishing
articles?) but in the rest of the United States, I can guarantee you there
will be some tissue in the Hematoxylin if you use a traditional dip and
dunk system.  

Liz, you do bring up a good point
about having tissue in every container. To a degree you
will have tissue in every reagent container. I was assuming and maybe
unjustly that most labs are using Good Laboratory Practice and
discard their reagents after they have used them for a staining session.  This
topic was about Hematoxylin Precipitate and "small spore or pollen-like
blue dots" which I did not say was tissue, I was passing along some
of my 23 years of knowledge.

Listen; don't take my word for
it.  You can have Ventana come out to your facility and filter
all your reagents and stains and have them tell you what is in your
solutions.
Next time just pass along good
information and criticize those trying to help!  To many people like to make a negative on this site of people trying to truly help.  This is why I rarely post on Histonet but instead directly email the person.

By no means am I promoting
Ventana/Roche products.  I am just passing along information that might be
helpful.  

 Tim Disclaimer: This information is by no means is meant for any weak stomach individuals or those preparing for the  zombie apocalypse.   

> From: gayle.callis at bresnan.net
> To: thigginsht at msn.com; histonet at lists.utsouthwestern.edu
> Subject: RE: [Histonet] Hematoxylin Precipitate
> Date: Tue, 22 Sep 2015 12:27:21 -0600
> 
> 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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