[Histonet] Hematoxylin Precipitate

Tue Sep 22 17:21:17 CDT 2015

Tim

First of all my comment was not meant to criticize your post and even if you may not think so I was trying to help.  I stand by what I said, my comment was to address what I thought was the amount of tissue loss your lab experiences and if I took your comment too literally I apologize but I firmly believe that properly processed and sectioned tissue samples should remain on the slide.  I do understand that we will on occasion have loss of tissue from the slides that we cut and stain.   We will see a lens floating in one of the alcohols when we stain mouse eyes or a portion of a dermal construct will come off the slides if we have not dried it properly.  Most of the tissue loss we experience is due to improperly processed and sectioned samples.   

We are a small lab and we are GLP compliant.  We do not change our H&E staining reagents daily our volume varies depending upon the projects we are working on, but I can tell you that we have looked at H&E staining over time and we have made sure that our reagents are changed appropriately.  We do not filter our hematoxylin daily and have not experienced many floaters or carry over or other things on our slides.  

Liz from wacky tabacky country

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
liz at premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

-----Original Message-----
From: Tim H via Histonet [mailto:histonet at lists.utsouthwestern.edu] 
Sent: Tuesday, September 22, 2015 3:04 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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