[Histonet] Re: Histonet Digest, Vol 29, Issue 6

From:"Robyn Vazquez"

it sounds like the paraffin is not melted in all the tissue sections.

Robyn
OHSU

>>> "Douglas Gondo"  4/5/2006 5:12 AM >>>
please can  anyone help I am histologist with a Bsc Uni. of Zimbabwe currently working for Namibia Institute of Pathology. my problem is I have seen that I am finding stained section that are not stained uniformly. Some areas are faint some dark as if there are holes in the sections. What could be the cause of this anormally?

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Today's Topics:

1. PTH (Richard Cartun)
2. Humedity chamber (cynthia haynes)
3. Fatty breast processing (Orr, Rebecca)
4. neutrophils (Webb, Dorothy L)
5. RE: neutrophils (Monfils, Paul)
6. Sirius red with low contrast between cells cytoplasm and
stained fibers (Guillermo Palao)
7. Re: Humedity chamber (Joanne Mauger)
8. RE: Thanks for the Input (Dave Low)
9. RE: Microtome knife sharpening service/ Microwave accessories
(Justin Thomas)
10. Humidity chamber (Donna Harclerode)
11. Histology Managers, Supervisors, and Bench Techs needed-
Intervirewing and Hiring NOW (Eric Dye (ext 223))
12. Hematoxylin staining of frozen samples, where are the nuclei?
(Guillermo Palao)
13. Qualifications for Histology Supervisor (Lester Raff)
14. Re: humidity chambers (Lori Richey)
15. Re: breast processing (Lori Richey)
16. Teaching Microtomes (Anwar Al-Banaw)
17. Re: Sirius red with low contrast between cells cytoplasm
andstained fibers (John Kiernan)
18. Feedback on Milestone Medical's RHS Series Microwave Tissue
Processor (jenbug812@aol.com)
19. RE: Sirius red with low contrast between cells cytoplasm
andstained fibers (Tony Henwood)
20. Re: Hematoxylin staining of frozen samples, where are the
nuclei? (Katri Tuomala)
21. Re: neutrophils (Jennifer MacDonald)
22. RE: Thanks for the Input (DDDeltour@mar.med.navy.mil)
23. RE: Thanks for the Input (Kemlo Rogerson)


----------------------------------------------------------------------

Message: 1
Date: Tue, 04 Apr 2006 13:13:52 -0400
From: "Richard Cartun" 
Subject: [Histonet] PTH
To: 
Message-ID: 
Content-Type: text/plain; charset=US-ASCII

Is anyone doing immunohistochemical staining for parathyroid hormone
(PTH) on formalin-fixed, paraffin-embedded tissue? If so, whose
antibody are you using? I just found out that DAKO no longer sells the
rat monoclonal antibody that we had been using.

It would be nice if suppliers notified their customers before they
discontinue a product so that we can find a replacement before we run
out of reagent.

Richard

Richard W. Cartun, Ph.D.
Director, Immunopathology & Histology
Assistant Director, Anatomic Pathology
Hartford Hospital
80 Seymour Street
Hartford, CT 06102
(860) 545-1596
(860) 545-0174 Fax







------------------------------

Message: 2
Date: Tue, 4 Apr 2006 11:10:55 -0700 (PDT)
From: cynthia haynes 
Subject: [Histonet] Humedity chamber
To: Histonet@lists.utsouthwestern.edu 
Message-ID: <20060404181055.97672.qmail@web33006.mail.mud.yahoo.com>
Content-Type: text/plain; charset=iso-8859-1

Hello all!
Some one put a website for a humedity chamber for
Immunohistochemistry staining. I think it was
www.scyetex.com. or something like that, would please
put the site address on the histonet again. Thanks in
advance.

Cynthia Haynes H.T.



------------------------------

Message: 3
Date: Tue, 4 Apr 2006 13:23:54 -0500
From: "Orr, Rebecca" 
Subject: [Histonet] Fatty breast processing
To: 
Message-ID:

Content-Type: text/plain; charset="US-ASCII"

HI Annette,
You have gotten quite a few responses to your question.
I have just one more. When we get huge pieces of fatty breast that is
unfixed, we place the block in the embedder, melt it and let it "soak"
in the paraffin for 2 hours. I have left them in this paraffin bath as
long as 4 hours but am unsure of any damage that could be done,( I
haven't seen any) But 2 seems to be as long as we need. I would think
that this process would cause damage, but since the tissue is unworkable
in the first place, this can only help.
This soaking in paraffin seems to do the trick, whether it's helping
infiltration or simply dehydrating, I imagine it's doing a little bit of
both.
After this process, the blocks are much easier to cut. I don't see any
problem with my IHC stains especially for ER/PR, Her2. These all seem
to stain consistently.
Ultimately, you would want to run your samples as others have
recommended before it gets to the embedding stage.

Hope this helps,
Becky


Becky Orr, CLA,HT(ASCP)
Assistant Manager, Anatomic Pathology
Evanston Northwestern Healthcare
847-570-2771

tm@libero.it>

Message: 5
Date: Tue, 4 Apr 2006 09:26:26 -0400
From: "Featherstone, Annette" 
Subject: [Histonet] breast processing
To: 
Message-ID:

<9B4A77DF11463E4FB723D484214AE9BCA55154@KALEXMB02.KaleidaHealth.org>
Content-Type: text/plain; charset="iso-8859-1"

Does anyone have any suggestions for better tissue processing for fatty
tissue such as breast? We are experiencing insufficent dehydration,
clearing and infiltration. 

Annette Featherstone HT/MLT





------------------------------

Message: 4
Date: Tue, 04 Apr 2006 13:25:14 -0500
From: "Webb, Dorothy L" 
Subject: [Histonet] neutrophils
To: Histonet@lists.utsouthwestern.edu 
Message-ID:
<0E394B648E5284478A6CCB78E5AFDA270171FD2A@hpes1.HealthPartners.int>
Content-Type: text/plain; charset="US-ASCII"

Does anyone know of a stain for neutrophils? I believe I had read that
staining with chloroacetate esterase is the way to go, but, I am not
familiar with that stain. Does anyone know of a company that would
have the stain in a kit?? This is for a research project and am rather
stumped! Thanks, as always, fellow Histonetters for your help and
support!!!!
________________________________________
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------------------------------

Message: 5
Date: Tue, 4 Apr 2006 14:37:46 -0400 
From: "Monfils, Paul" 

Subject: RE: [Histonet] neutrophils
To: "'histonet@lists.utsouthwestern.edu'" 

Message-ID:
<09C945920A6B654199F7A58A1D7D1FDE017176BC@lsexch.lsmaster.lifespan.org>

Content-Type: text/plain; charset="iso-8859-1"

I use the Naphthol AS-D Chloroacetate Esterase Kit from Sigma (cat# 91C-1KT)
on frozen sections. It's easy to use and very reliable.

> ----------
> From: histonet-bounces@lists.utsouthwestern.edu on behalf of Webb,
> Dorothy L
> Sent: Tuesday, April 4, 2006 11:25 AM
> To: Histonet@lists.utsouthwestern.edu 
> Subject: [Histonet] neutrophils
> 
> Does anyone know of a stain for neutrophils? I believe I had read that
> staining with chloroacetate esterase is the way to go, but, I am not
> familiar with that stain. Does anyone know of a company that would
> have the stain in a kit?? This is for a research project and am rather
> stumped! Thanks, as always, fellow Histonetters for your help and
> support!!!!
> ________________________________________
> This e-mail and any files transmitted with it are confidential and are
> intended solely for the use of the individual or entity to whom they are
> addressed. If you are not the intended recipient or the individual
> responsible for delivering the e-mail to the intended recipient, please be
> advised that you have received this e-mail in error and that any use,
> dissemination, forwarding, printing, or copying of this e-mail is strictly
> prohibited.
> 
> If you have received this e-mail in error, please immediately notify the
> HealthPartners Support Center by telephone at (952) 967-6600. You will be
> reimbursed for reasonable costs incurred in notifying us.
> _______________________________________________
> Histonet mailing list
> Histonet@lists.utsouthwestern.edu 
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet 
> 
> 



------------------------------

Message: 6
Date: Tue, 4 Apr 2006 20:58:33 +0200 (CEST)
From: Guillermo Palao 
Subject: [Histonet] Sirius red with low contrast between cells
cytoplasm and stained fibers
To: Histonet 
Message-ID: <20060404185833.93523.qmail@web26202.mail.ukl.yahoo.com>
Content-Type: text/plain; charset=iso-8859-1

Hello all:

I would really appreciate any suggestions to improve sirius red staining of kidney samples. Although collagen fibers in tissue nicely stain red, cells cytoplasm do not stain yellow, but sort of pinkish, making it very difficult to separate from red-stained extracellular fibers for quantification purposes.

My protocol is as follows:

1. Deparafinize and hydrate samples
2. Stain 1 h in 0.1% Sirius Red in saturated aqueous picric acid
3. Wash 2 min 0.01 N HCl
4. Rinse in water
5. Dehydrate and cover slides

I have tried shorter staining times and rinsing with water before HCl washing without improving results. Any help would be greatly appreciated.

Guillermo


---------------------------------

LLama Gratis a cualquier PC del Mundo.
Llamadas a fijos y móviles desde 1 céntimo por minuto.
http://es.voice.yahoo.com 

------------------------------

Message: 7
Date: Tue, 04 Apr 2006 15:49:36 -0400
From: "Joanne Mauger" 
Subject: Re: [Histonet] Humedity chamber
To: ,
Message-ID: 
Content-Type: text/plain; charset=US-ASCII

Cynthia,

It's www.scytek.com 

Jo

>>> cynthia haynes 04/04/06 2:10 PM >>>
Hello all!
Some one put a website for a humedity chamber for
Immunohistochemistry staining. I think it was
www.scyetex.com. or something like that, would please
put the site address on the histonet again. Thanks in
advance.

Cynthia Haynes H.T.

_______________________________________________
Histonet mailing list
Histonet@lists.utsouthwestern.edu 
http://lists.utsouthwestern.edu/mailman/listinfo/histonet 



------------------------------

Message: 8
Date: Tue, 4 Apr 2006 13:19:41 -0700 (PDT)
From: Dave Low 
Subject: RE: [Histonet] Thanks for the Input
To: DDDeltour@mar.med.navy.mil, vazquezr@ohsu.edu,
Jackie.O'Connor@abbott.com, Heather.A.Harper@pcola.med.navy.mil 
Cc: histonet@lists.utsouthwestern.edu 
Message-ID: <20060404201941.10561.qmail@web32004.mail.mud.yahoo.com>
Content-Type: text/plain; charset=iso-8859-1

Douglas,

Military personnel DO NOT make rank by working at some
bake sales! Are you insinuating Master Chief Petty
Officers, Sergeant Majors, and Chief Master Sargeants
made rank selling cookies? Rubbish!

I am an Air Force Master Sargeant, worked in histology
for over 17 years out of my 20+ years service. I
worked at the Armed Forces Institute of Pathology in
Washington DC from 1997-2000 and had the pleasure to
work for the Army, Navy, and civilians histology
technicians and managers. The promotions I did see
were from hard work not only from the job but to the
military and civilian communities. It's called the
whole person concept!

In the future please screen your e-mail for
appropriateness before you broadcast to a large
audience like histonet. 

Dave Low, MSgt,USAF
HT(ASCP)QIHC

--- DDDeltour@mar.med.navy.mil wrote:

> I work in this environment everyday. I see both
> sides (military and
> civilian) taking shots at each other instead of
> working as a team. I am soon
> leaving the service and you could not pay me enough
> money to supervise or
> work in a military facility. I understand Heather
> and her frustration of
> being left to do the job BUT the military requires
> its members to do things
> outside of the job to get promoted. That is a fact.
> I am living it. I see
> people in my own field get promoted because they did
> some BS bake sale but
> they can't even do a GMS. You can either find a way
> to work it out or find a
> new job. As for the military Pathologist....well
> they are in charge. They
> like that. Everything will change again when the
> next one transfers in. That
> is part of being in a military facility. I have seen
> it for years. The best
> thing to do is go with the flow or just go. I am
> going. Good luck. 
> 
> Douglas D. Deltour HT(ASCP)
> 
> 
> -----Original Message-----
> From: histonet-bounces@lists.utsouthwestern.edu 
> [mailto:histonet-bounces@lists.utsouthwestern.edu] 
> On Behalf Of Robyn
> Vazquez
> Sent: Monday, April 03, 2006 12:29 PM
> To: Jackie.O'Connor@abbott.com; Harper, Heather A.,
> CIV
> Cc: histonet@lists.utsouthwestern.edu 
> Subject: Re: [Histonet] Thanks for the Input
> 
> Sounds like you both work hard...I have been on both
> sides of the
> fence...keep up the good work...she needs all the
> support you can give her
> civilian or military!
> Just my two cents...
> 
> Robyn
> OHSU
> 
> >>> "Jackie M O'Connor" 
> 4/3/2006 8:49 AM >>>
> I am thankful and grateful for anyone who ever
> signed up for military 
> service - ever - regardless of their job
> description.
> 
> 
> 
> 
> 
> Heather.A.Harper@pcola.med.navy.mil 
> Sent by: histonet-bounces@lists.utsouthwestern.edu 
> 04/03/2006 10:20 AM
> 
> 
> To: histonet@lists.utsouthwestern.edu 
> cc: (bcc: Jackie M
> O'Connor/LAKE/GPRD/ABBOTT)
> Subject: [Histonet] Thanks for the
> Input
> 
> 
> I want to thank everybody who responded to my
> message titled..Need Input. 
> I
> work for the DOD and I miss simply having a system
> in place. I agree one 
> has
> to be flexible, but when you work with military,
> they are entitled to 1 hr
> lunch and 1 hour of PT. My tech works 7-4 and I work
> 6-2. I embed, she 
> cuts
> and stains, I gross, accession, order supplies,
> admit bodies in and out of
> the morgue, set up for autopsies, frozens, and when
> my tech has to get
> pulled to do her other command duties, it leaves me
> holding the bag. It
> does get over whelming, and every 2-3 yrs, I get new
> pathologists rotating
> through. I have worked with 7 pathologists in 6 yrs,
> and reservists, and
> this one I just do not know what to think. Just
> keeping an open mind. 
> Thanks
> again everybody. Be thankful you are in the civilian
> world.
> 
> 
> 
> Heather
> 
> 
> 
> _______________________________________________
> Histonet mailing list
> Histonet@lists.utsouthwestern.edu 
>
http://lists.utsouthwestern.edu/mailman/listinfo/histonet 
> 
> 
> 
> _______________________________________________
> Histonet mailing list
> Histonet@lists.utsouthwestern.edu 
>
http://lists.utsouthwestern.edu/mailman/listinfo/histonet 
> 
> 
> 
> 
> 
> _______________________________________________
> Histonet mailing list
> Histonet@lists.utsouthwestern.edu 
>
http://lists.utsouthwestern.edu/mailman/listinfo/histonet 
> _______________________________________________
> Histonet mailing list
> Histonet@lists.utsouthwestern.edu 
>
http://lists.utsouthwestern.edu/mailman/listinfo/histonet 
> 


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------------------------------

Message: 9
Date: Tue, 4 Apr 2006 13:22:18 -0700 (PDT)
From: Justin Thomas 
Subject: [Histonet] RE: Microtome knife sharpening service/ Microwave
accessories
To: histonet@lists.utsouthwestern.edu 
Message-ID: <20060404202218.93567.qmail@web35701.mail.mud.yahoo.com>
Content-Type: text/plain; charset=iso-8859-1

Just to let everyone know:

There is finally a company out there that sharpens microtome knifes 
(both stainless steel & tungsten) and manufactures plastic microwave 
accessories at a low price. I send all our knifes to them and I am 
extremely satisfied with the customer service I receive, along with the 
pricing. Also, they have made a number of accessories for us and I have 
nothing but good words to say about them. The accessory's work well and 
with no problem. The best thing about them is the company offers a 
warranty with each accessory. If you wish, email me back with your contact 
information and I will pass it on to the sharpening and MW accessory 
company.

Thanks,
Dr. Thomas


---------------------------------
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------------------------------

Message: 10
Date: Tue, 4 Apr 2006 13:36:57 -0700
From: "Donna Harclerode" 
Subject: [Histonet] Humidity chamber
To: 
Message-ID:
<3DE0F644E093DF4BAE80C254176696A5090706@mp-mailserver.macropore.com>
Content-Type: text/plain; charset="us-ascii"

Thermo Electron makes a great system with a humidity chamber and a
coverplate that I have used for many years. The slides are held with
the plastic coverplate(the coverplates are suppose to be disposable, but
I rinse them with only DI water and reuse them many times) I use 100ul
of antibody (primary, secondary- etc, normally, but have managed to use
as little as 90ul) to cover almost the whole slide. For buffer wash I
fill up the top of the chamber with a squeeze bottle of PBS and allow to
run through in about 5 minutes. I load the slides add primary, wash,
secondary etc and only take them out for chromagen staining.
The racks worked great when I had the positive control at the top of the
slide and the test below. I do fluorescence secondaries in the
coverplates, but I not do DAB in the coverplates, but flat on the
counter. 
For HRP frozens I use the Dako endogenous peroxidase block in the
Sequenza racks. I also use the Dako avidin biotin block in the
coverplates when necessary. 
I usually incubate primary overnight in the fridge and the chambers stay
humid for at least 48 hours. It takes a bit of practice to load the
plates, but it is so worth it.

Sequenza Racks - # 73310017 (Holds 10 coverplate assemblies)

Coverplates - 25/pk = # 72110017
50/pk = # 7219950
250/case = # 72110013
http://www.thermo.com/eThermo/CMA/PDFs/Product/productPDF_19578.pdf 4th
page


Donna Harclerode, HT, (ASCP), HTL, QIHC
Scientist / Immunohistochemistry
Cytori Therapeutics
3020 Callan Rd.
San Diego, CA 92121
858-458-0900 ext 5416
dharclerode@cytoritx.com 



------------------------------

Message: 11
Date: Tue, 4 Apr 2006 17:13:06 -0400
From: Eric Dye (ext 223) 
Subject: [Histonet] Histology Managers, Supervisors, and Bench Techs
needed- Intervirewing and Hiring NOW
To: Histonetters 
Message-ID: 
Content-Type: text/plain

Fellow-Histonetters
I'm presently on a search for my best client companies seeking HistoTech Supervisors and Histo Bench Techs. These are mostly fulltime permanent positions. Most of my Histo Techs positions are clinical, although I do have a few Temporary and Research Histo Tech positions as well. Most Histo jobs are dayshift (Except where indicated below)

Here are some of my Hottest Histology Jobs:

1. Ohio (Southwestern Ohio) (Full-time, Perm, Histo Manager)

2. Northern New Jersey (Full-time, Perm and/or Temp, Bench Histo Tech, 1st shift)

3. Rhode Island - Providence (Full-time, Perm, Bench Histo Tech, 2nd Shift)

4. Northern New Jersey (Full-time, Perm, Bench Histo Tech)

5. Ohio (Southwestern Ohio) (Full-time, Perm, Bench Histo Tech)

6. New York (Long Island) (Full-time, Perm, SUPERVISOR Histo Tech)

7. Virginia(South - Coastal) (Full-time, Perm, Bench Histo Tech)


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