[Histonet] RE: Histonet Digest, Vol 27, Issue 2 IHC Help! (Heckford, Karen - SMMC-SF)

From:Niclas Bjorkhammer

Hi,

There are several antibodies that can aid the differentiation between
microglandular adenosis and other conditions of the breast. Although
Collagen IV has shown interesting results, I personally prefer the SMA or
S-100, as these will highlight the BM better than with Collagen IV. Having
said that, there have been discussions relating to the use of SMA , due to
the extensive cross reaction of SMA with myofibroblasts, (smooth muscle
myosin heavy chains and calponin). Probably more promising is the study
showing the presence of an intensely positive reaction in the epithelial
lining cells of the tubules in microglandular adenosis for S-100 protein.
Might be worth a try.

There is a good article published in 1996 that discusses the various
immunohistochemical markers.

The role of immunocytochemical markers in the differential diagnosis of
proliferative and neoplastic lesions of the breast.

Mod Pathol. 1996 Jan;9(1):57-62.


Niclas Björkhammer
Senior Biomedical Scientist
Department of Pathology, Histopathology
Weston General Hospital
Weston Area Health Trust
01934 647 056 ext 3313
carl.bjorkhammer@waht.swest.nhs.uk


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To: histonet@lists.utsouthwestern.edu
Subject: Histonet Digest, Vol 27, Issue 2

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Today's Topics:

   1. RE: processing  (Gayle Callis)
   2. collecting fresh tissue prior to perfusion (Caroline Bass)
   3. is Methyl Green suitable for water-based mounting media? (andres)
   4. Hospitals that  use Temps (Orr, Rebecca)
   5. Bone/ Cartilage Interface (Sheffield, Tiffany L)
   6. Re: Hospitals that  use Temps (Cheryl)
   7. IHC Help! (Heckford, Karen - SMMC-SF)
   8. looking for Antibodies-MSH6  and PMS2 (Nava, Josefa)
   9. Re: Active caspase 3 staining of mouse small intestine
      (John C. Dennis)
  10. Bizarre Immunofluorescence problem in mouse brain	sections
      (Anna Elisse Beaudin)
  11. Tissue Processor Paraffin Temps for CAP Inspections (Amy Porter)
  12. Re: Tissue Processor Paraffin Temps for CAP Inspections
      (Rene J Buesa)
  13. RE: Tissue Processor Paraffin Temps for CAP Inspections
      (Joe Nocito)
  14. Re: Tissue Processor Paraffin Temps for CAP  Inspections
      (LuAnn Anderson)
  15. RE: Active caspase 3 staining of mouse small intestine
      (Goodpaster, Tracy A)
  16. citation for negative controls (Jonathan Wilson)
  17. RE: citation for negative controls (Tony Henwood)
  18. RE: processing  (Kemlo Rogerson)
  19. RE: Tissue Processor Paraffin Temps for CAP Inspections
      (Molinari, Betsy)
  20. Kuppfer cells (Susan.Ferrigon@sanofi-aventis.com)
  21. microwave transparent containers and equipment (Adams, Nancy)
  22. Re: Kuppfer cells (GT Hebert)
  23. Re: microwave transparent containers and equipment (Rene J Buesa)
  24. FW: [Histonet] Tissue Processor Paraffin Temps for CAP
      Inspections (Bruijntjes, J.P.)
  25. Re: citation for negative controls (Gayle Callis)


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

Message: 1
Date: Wed, 01 Feb 2006 11:35:14 -0700
From: Gayle Callis 
Subject: RE: [Histonet] processing 
To: Kemlo Rogerson ,
	Histonet@lists.utsouthwestern.edu
Message-ID:
	<6.0.0.22.1.20060201111051.01b4f290@gemini.msu.montana.edu>
Content-Type: text/plain; charset="us-ascii"; format=flowed

You are not rambling.   Animal tissues, particularly rodent are very lean, 
and if one really wants to see dry animal tissue, tissues from birds of 
prey (hawks), other birds and reptiles can be among the worst. We custom 
process our rodent tissues, brain, small, etc and have shorter schedules 
for mouse brain versus hamster brain, that type of thing just to avoid 
overdehydration.

The 1 hour per station or change even with 3 X 95% and 3 x 100% never seems 
to bother human species tissues much, but with rodent tissue, it removes 
too much of the bound water.  some larger animals with bigger samples in a 
cassette seem to be ok with standard processing schedules but never with 
heat added.

A curious thing observed over a long career in histowork, I saw the 
addition of heat to processing in automated processor a curious 
thing.  When we used the older carousel (Sp?)  models i.e Technicon, heat 
couldn't be added and processing was done at RT.  True, heat may speed up 
the solvent exchange but it also can add to drying of tissues.  I have 
always wondered why this has become a standard method over the years other 
than trying to speed up processing, but always felt alternating vacuum and 
pressure to be the best way to do that along with paying attention to 
length of processing schedules.

What one needs to do is find the correct balance so that the free water in 
the tissue spaces is removed and not the bound water on protein 
molecules.  Overdehydration and adding heat to processing will exacerbate 
the removal of bound water. Consequently, heat is never added to our animal 
tissue processing steps.




  At 02:38 AM 2/1/2006, you wrote:
>Think rodent tissue can process on the 'hard' side. Wonder what makes it
>'hard'? Is it that the bonds between the proteins are 'stronger' or the
>proteins are 'nearer' together? I mean if we could figure out what made
>tissue 'hard' molecularly then we'd know which the culprit in the
processing
>was.
>
>Sorry to ramble.
>
>Kemlo Rogerson
>Pathology Manager
>Ext  3311
>DD   01934 647057
>Mob 07749 754194
>
>
>
>
>-----Original Message-----
>From: Steven Coakley [mailto:sjchtascp@yahoo.com]
>Sent: Tuesday, January 31, 2006 6:56 PM
>To: Histonet@lists.utsouthwestern.edu
>Subject: [Histonet] processing
>
>Good afternoon,
>
>   I'm working with mouse/rat muscle and trying to fine tune my processing
>schecdule.
>   My last run of muscle were very dry.  I have a copy of the NSH Animal
>Processing Manual and trying to strike a balance between those listed on
>page 5-6.  I've noticed most of the protocols do not call for heat or P/V.
>The scedule I currently use is as follows: 1-70% (hold), 1-80%, 2-95%,
>3-100%, 3-xylene all for 30 minutes each and at 38C with P/V.  3 paraffins,
>45 minutes each at 55C with P/V.  I'm fairly new to research and still
>trying to make the "mental" switch in handling the different tissue.
>
>   Thanks everyone,
>
>   Steve
>
>
>
>---------------------------------
>Bring words and photos together (easily) with
>  PhotoMail  - it's free and works with Yahoo! Mail.
>_______________________________________________
>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

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)





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

Message: 2
Date: Wed, 1 Feb 2006 13:52:03 -0500
From: Caroline Bass 
Subject: [Histonet] collecting fresh tissue prior to perfusion
To: Histonet (E-mail) 
Message-ID: 
Content-Type: text/plain; charset=US-ASCII; delsp=yes; format=flowed

Hello,

I have what may be a silly question but I thought maybe it is worth a  
try.  I am in a situation where the best way to collect liver tissue  
for my purposes is to perfuse the mouse with PFA.  However, on  
occasion I need a small bit of fresh liver tissue to analyze for  
DNA.  I was wondering if it is possible to collect a portion of a  
liver lobe at some point in the perfusion process before I use  
fixative?  For example, I could start the perfusion with heparinized  
saline, clip a quarter of one lobe of the liver off and then switch  
over to the PFA.  I would then proceed with the perfusion as normal.

The only complication I can think of is that the loss of a  
significant portion of the liver could effect how well the rest of  
the liver perfuses.

Has anyone tried this or is this something I should avoid?  I am  
trying to cut down on the number of mice I am using.

My goal is to obtain a completely fixed liver lobe that I can section  
all the way through.  It is my understanding that the mouse liver is  
too large to fix by immersion in PFA, so it either has to be cut into  
blocks or the mouse perfused.

Thanks,

Caroline

  



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

Message: 3
Date: Wed, 01 Feb 2006 19:55:36 +0100
From: andres 
Subject: [Histonet] is Methyl Green suitable for water-based mounting
	media?
To: 
Message-ID: <6.2.1.2.0.20060201194542.02840978@localhost>
Content-Type: text/plain;	charset=iso-8859-1;	format=flowed



Hi everybody,

I'm trying to counterstain my in situ hybridizations with methyl green and 
want to mount them in glycerol or similar.  I'd like to ask if any of you 
have done (or if it washes away with time...since all the protocols I've 
seen are followed by dehydration prior to mounting).

Also, since I can't get here the already prepared solution and I've seen 
several ways to prepare the staining solution from powder(Aldrich) any 
suggestion about how to do it??


Thanks a lot !



Andrés Kamaid

Dpto. Histología
Facultad de Medicina
Universidad de la República
Montevideo - Uruguay




At 17:04 01/02/2006, James Watson wrote:
>Steve,
>
>I routinely use 5% glycerin in the absolute alcohols for my mouse 
>tissues.  this has shortened our soaking times and eliminated the drying 
>artifact.  I started using this years ago on whole cross sections of dog 
>hearts that were processed at a 2.5 cm thickness and on frog leg muscle 
>that was very dry.  I am in the process of completeing some comparative 
>studies and hope to get this published soon.  if you want my processing 
>schedules let me know.
>
>James Watson
>GNF San Diego
>jwatson@gnf.org
>
>________________________________
>
>From: histonet-bounces@lists.utsouthwestern.edu on behalf of Steven Coakley
>Sent: Tue 1/31/2006 10:55 AM
>To: Histonet@lists.utsouthwestern.edu
>Subject: [Histonet] processing
>
>
>
>Good afternoon,
>
>   I'm working with mouse/rat muscle and trying to fine tune my processing 
> schecdule.
>   My last run of muscle were very dry.  I have a copy of the NSH Animal 
> Processing Manual and trying to strike a balance between those listed on 
> page 5-6.  I've noticed most of the protocols do not call for heat or 
> P/V.  The scedule I currently use is as follows: 1-70% (hold), 1-80%, 
> 2-95%, 3-100%, 3-xylene all for 30 minutes each and at 38C with P/V.  3 
> paraffins, 45 minutes each at 55C with P/V.  I'm fairly new to research 
> and still trying to make the "mental" switch in handling the different
tissue.
>
>   Thanks everyone,
>
>   Steve
>
>
>
>---------------------------------
>Bring words and photos together (easily) with
>  PhotoMail  - it's free and works with Yahoo! Mail.
>_______________________________________________
>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




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

Message: 4
Date: Wed, 1 Feb 2006 13:06:41 -0600
From: "Orr, Rebecca" 
Subject: [Histonet] Hospitals that  use Temps
To: 
Message-ID:
	
Content-Type: text/plain;	charset="us-ascii"

Hi Everyone,
We are considering using a temp  agency to cover some short leaves and
maybe fill in PRN. 
What do you do with training and competency that needs to be done to
fulfill JCAHo?
What about the in-house hospital training? Do you adjust your training
to accommodate these temps?
In your experience, are the temp agencies  responsible for some of this
training?

We are thinking a temp would need the same training as our staff.

Does anyone have experience they would like to share? I'd appreciate it.

Thank  you
Becky

 Becky Orr CLA,HT(ASCP)
IHC Lead 
Evanston Northwestern Healthcare
847-570-2771
 





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

Message: 5
Date: Wed, 1 Feb 2006 13:29:03 -0600
From: "Sheffield, Tiffany L" 
Subject: [Histonet] Bone/ Cartilage Interface
To: 
Message-ID:
	
Content-Type: text/plain;	charset="us-ascii"

Quick question-
Has anyone done any Ab staining of the bone cartilage interface? If you
have and can share the type of Ab and/or tips it would be greatly
appreciated.
Thanks!
 
Tiffany Sheffield-Lopez, HT(ASCP)
Supervisor, Bone Histomorphometry & Biomaterials Lab
Department of Orthopaedic Surgery
UTHSC-Houston Medical School
6431 Fannin, Suite 6.144 MSB
Houston , TX 77030
713-500-6803 WK
713-500-0729 Fax
 


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

Message: 6
Date: Wed, 1 Feb 2006 11:49:25 -0800 (PST)
From: Cheryl 
Subject: Re: [Histonet] Hospitals that  use Temps
To: "Orr, Rebecca" , histonet@lists.utsouthwestern.edu
Message-ID: <20060201194925.21945.qmail@web50907.mail.yahoo.com>
Content-Type: text/plain; charset=us-ascii

Hi Becky-
 
I operate a Histo/Path Anatomy placement agency (permanent staffing only but
have done temp services) so let's see if I can help you with your current
situation:
 
You are obligated to have them trained in all the things JCAHO requires (all
those for which you train your perm employess)  and you need to have a file
for each one in the same place you keep your regular employee records.
There are different ways to get there.  Some of the bigger agencies conduct
this training by video and online reading when they contract the tech.  Talk
to your recruiting contact at each agency.  Some of these people have been
at other facilities as permanents and can provide proof from their own
records or by requesting copies from their last employer.  Rarely an
ex-employer won't share these records putting you back at square one.  Be
careful with dates if these are annual training situations like safety and
BBPathogens.  
 
Most facilities that regularly use temps bring them in on the hiring cycle
and put them through a full orientation.  You DO pay for the tech's time but
it's far more cost effective than a big hit from JCAHO or any of the other
governing bodies requiring safety, BBPathogens, etc. If your HR has a
mini-orientation that allows an employee to start 'off-cycle' but covers all
the bases then that is the best way to cover all your requirements in the
most cost-effective manner.  If your HR contact isn't sure and you have a
nurse recruiter in your facility, this person will probably know in one
phone call what you need to do to get your new temps up to speed.
 
Please consider that for some of this, training is site specific, and the
care with which you orient these folks will come back to you in many ways as
they will be better temps knowing you care enough to train them correctly
and well.  I temped on and off for over ten years and there are labs where I
stayed as a permanent because I was well treated and loved the job.  Most
temps are consciencious professionals and great workers.  I hope you've been
staffed with several of these as they really contribute and get you through
the shortage with grace. 
 
Please let me know if you are seeking permanent solutions in your facility.
Our services are a fraction of the other agencies--I have a regular job and
helping good techs and great labs find each other is a 'calling'  -- I've
love to help.
 
Cheryl Kerry
Full Staff Inc
281.883.7704

----- Original Message ----
From: "Orr, Rebecca" 
To: histonet@lists.utsouthwestern.edu
Sent: Wednesday, February 01, 2006 1:06:41 PM
Subject: [Histonet] Hospitals that use Temps


Hi Everyone,
We are considering using a temp  agency to cover some short leaves and
maybe fill in PRN. 
What do you do with training and competency that needs to be done to
fulfill JCAHo?
What about the in-house hospital training? Do you adjust your training
to accommodate these temps?
In your experience, are the temp agencies  responsible for some of this
training?

We are thinking a temp would need the same training as our staff.

Does anyone have experience they would like to share? I'd appreciate it.

Thank  you
Becky

Becky Orr CLA,HT(ASCP)
IHC Lead 
Evanston Northwestern Healthcare
847-570-2771




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

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

Message: 7
Date: Wed, 1 Feb 2006 12:53:07 -0700
From: "Heckford, Karen - SMMC-SF" 
Subject: [Histonet] IHC Help!
To: "Histonet (E-mail)" 
Message-ID:
	
Content-Type: text/plain; charset=iso-8859-1

Dear Histonetters,  
One of my pathologists needs a antibody that will show microglandular
adenosis in human breast tissue.  I am working with FFPE tissue and the Dako
Autostainer.  I have tried Neomarkers Collagen IV Ab-3 with Dako's Envision
system.  At the time I did not have any Protease so I used Protinease K and
later used the Protease.    I did get my controls to come back positive and
my negative were negative all three times,  but the patient tissue did not
show microglandular adenosis.  We did sent this tissue out and it came back
positive for microglandular adenosis.  I cannot figure out what I am doing
wrong or perhaps I bought the wrong antibody.   

I would really appreciate any help.  This has been bothering me for a week
now.

Karen Heckford HT (ASCP) CE
kheckfor@chw.edu
1-415-668-1000 ext. 6167




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

Message: 8
Date: Wed, 1 Feb 2006 13:54:36 -0600
From: "Nava, Josefa" 
Subject: [Histonet] looking for Antibodies-MSH6  and PMS2
To: 
Message-ID:
	<2C515C1049EAF5459EFD8C9B929078A41945A8@phdex03.txhealth.org>
Content-Type: text/plain;	charset="us-ascii"

Hello Everyone ,
I am using Ventana BMK/XT , can someone tell me a good source of MSH6
and PMS2  that will work  on the Ventana  Machine. I  appreciate  any
information. Thank you.
 
Josie 
 


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

Message: 9
Date: Wed, 1 Feb 2006 14:38:37 -0600 (CST)
From: "John C. Dennis" 
Subject: Re: [Histonet] Active caspase 3 staining of mouse small
	intestine
To: "Yu, Jian" 
Cc: histonet@lists.utsouthwestern.edu
Message-ID: 
Content-Type: TEXT/PLAIN; charset=US-ASCII; format=flowed


I'm preparing a Caspase 3 preparation today, in fact.  The tissue is 
paraffin embedded rat ethmoturbinate.  I boil the slides in 10 mM Na 
Citrate 
pH 6.0 20'.  I'm using Sigma's rabbit polyclonal and Vector's DAB kit.

John Carroll Dennis
Anatomy, Physiology, and Pharmacology
109 Greene Hall
Auburn University, AL  36849


On Wed, 1 Feb 2006, Yu, Jian wrote:

> Thanks to several of you who have given me a lot of great suggestions on
> how to get good cross sections of mouse small intestine   One of you
> mentioned that you do Caspase 3 staining routinely with the small
> intestine, sorry that I could not find your email anyone.  I have been
> using the CAM1 Ab from BD, which works well for IF on frozen sections
> but have a lot of background for IHC.  Unfortunately the frozen sections
> do not have the greatest structures.  I would very much appreciate that
> if you could share your experience with paraffin sections.
>
>
>
> Thanks again!
>
> ********************************************************
>
> Jian Yu, Ph. D.
>
> University of Pittsburgh Cancer Institute
>
> Hillman Cancer Center Research Pavilion
>
> Office suite 2.26h, Laboratory 2.43
>
> 5117 Centre Avenue, Pittsburgh, PA 15213
>
>
>
> Phone : 412-623-7786, (Lab) 412-623-3255
>
> Fax:      412-623-7778
>
> Email:   yuj2@upmc.edu
>
> ********************************************************
>
>
>
> _______________________________________________
> Histonet mailing list
> Histonet@lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>



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

Message: 10
Date: Wed, 1 Feb 2006 15:58:12 -0500 (EST)
From: "Anna Elisse Beaudin" 
Subject: [Histonet] Bizarre Immunofluorescence problem in mouse brain
	sections
To: histonet@lists.utsouthwestern.edu
Message-ID:
	<1506.128.253.96.73.1138827492.squirrel@webmail.cornell.edu>
Content-Type: text/plain;charset=iso-8859-1

Dear Histonet,

   I am having a bizarre problem with an immunofluorescence protocol that
I am really hoping someone can help me with.  here are the specs:

I'm doing immunofluorescence for BrdU in PFA-fixed mouse brain sections
that have been collected in PBS, mounted on slides, and allowed to dry 2+
days. Once dried, slides are processed for IHC as follows:
Antigen retrieval (20min in  .1M citrate buffer pH6.0, 95C)
2N HCl treatment
10% normal serum block followed by 10% casein block
rat primary incubation O/N at 4C
5min rinse with PBS, .1% triton X, + 2X3 min PBS
Secondary is a jackson Aexa488-conjugated goat anti-rat - 45 min at room
temp
Rinses... coverslip

The problem I am encountering is the appearance of strange fiber-like
pieces on my tissue that are picking up the secondary.  at 40X they look
like segmented strings or fibers (almost like bacteria). I have no idea
where they're coming from.  I did NOT have this problem with regular HRP
development, and my secondary is brand new.  I have this same problem even
with a rat IgG control.  I am still getting good specific staining, I just
need to get rid of this stringy background.  Does anybody have any
suggestions/ideas about what this is and how I might get rid of it?  I
really appreciate your help.

Best,
Anna Beaudin
Division of Nutritional Sciences
Cornell University





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

Message: 11
Date: Wed, 1 Feb 2006 16:12:54 -0500
From: "Amy Porter" 
Subject: [Histonet] Tissue Processor Paraffin Temps for CAP
	Inspections
To: "Histonet" 
Message-ID: <001a01c62774$44c5ef70$8e7a0923@HistoJJ>
Content-Type: text/plain;	charset="iso-8859-1"

If your willing to share - how are people monitoring the temperature of
paraffin baths on tissue processors?  Are you using additional thermometers
directly into the paraffin baths, or just going by the digital read out on
the display for the (oven on a VIP) instrument?  Just curious how everyone
is dealing with this particular item.  Thanks in advance for the many
responses and ideas I know I will get from asking this question!
Amy S. Porter, HT(ASCP) QIHC
Laboratory Supervisor
Michigan State University
Investigative HistoPathology Laboratory 
Department of Physiology / Division of Human Pathology
2100 Biomedical Physical Sciences Bldg. Room #2133
East Lansing, MI  48824-3320
Phone:  (517) 355-6475 ext 1480 / Fax:  (517) 432-1368
Email:  portera@msu.edu
www.humanpathology.msu.edu



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

Message: 12
Date: Wed, 1 Feb 2006 13:22:01 -0800 (PST)
From: Rene J Buesa 
Subject: Re: [Histonet] Tissue Processor Paraffin Temps for CAP
	Inspections
To: Amy Porter , Histonet
	
Message-ID: <20060201212201.1914.qmail@web61215.mail.yahoo.com>
Content-Type: text/plain; charset=iso-8859-1

Amy:
  The digital thermometer in the VIP is not only very accurate but the most
expeditious way of checking the temperature.
  Just prepare a log that include all your VIPs and record the temperature
once a day.
  That is the way I did it, and never had problems with CAP inspectors.
  Hope this will help you.
  René J.

Amy Porter  wrote:
  If your willing to share - how are people monitoring the temperature of
paraffin baths on tissue processors? Are you using additional thermometers
directly into the paraffin baths, or just going by the digital read out on
the display for the (oven on a VIP) instrument? Just curious how everyone is
dealing with this particular item. Thanks in advance for the many responses
and ideas I know I will get from asking this question!
Amy S. Porter, HT(ASCP) QIHC
Laboratory Supervisor
Michigan State University
Investigative HistoPathology Laboratory 
Department of Physiology / Division of Human Pathology
2100 Biomedical Physical Sciences Bldg. Room #2133
East Lansing, MI 48824-3320
Phone: (517) 355-6475 ext 1480 / Fax: (517) 432-1368
Email: portera@msu.edu
www.humanpathology.msu.edu

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




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

Message: 13
Date: Wed, 1 Feb 2006 15:30:05 -0600
From: "Joe Nocito" 
Subject: RE: [Histonet] Tissue Processor Paraffin Temps for CAP
	Inspections
To: "'Amy Porter'" ,	"'Histonet'"
	
Message-ID:
	
Content-Type: text/plain;	charset="US-ASCII"

Amy,
We take the temps of each individual bath, but beware. During one of my
inspections, an inspector had his own thermometer and tested the temps.
Although we had the temps at 60C, the baths read 65C. This was out of our
range and he wrote me up. I called Sakura tech rep and had them explain to
the inspector why the temp difference. Tech support told the inspector that
the baths were hotter to account for the paraffin cooling off during
transfer to the retort chamber. He bought it. 
	And people wonder why I have a negative attitude about CAP. How many
people have been inspected by an inspector who carries his/her own
thermometer?  I should have asked him to show me documentation that it was
calibrated.

Joe "The Toe" Nocito

-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu
[mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of Amy Porter
Sent: Wednesday, February 01, 2006 3:13 PM
To: Histonet
Subject: [Histonet] Tissue Processor Paraffin Temps for CAP Inspections

If your willing to share - how are people monitoring the temperature of
paraffin baths on tissue processors?  Are you using additional thermometers
directly into the paraffin baths, or just going by the digital read out on
the display for the (oven on a VIP) instrument?  Just curious how everyone
is dealing with this particular item.  Thanks in advance for the many
responses and ideas I know I will get from asking this question!
Amy S. Porter, HT(ASCP) QIHC
Laboratory Supervisor
Michigan State University
Investigative HistoPathology Laboratory 
Department of Physiology / Division of Human Pathology
2100 Biomedical Physical Sciences Bldg. Room #2133
East Lansing, MI  48824-3320
Phone:  (517) 355-6475 ext 1480 / Fax:  (517) 432-1368
Email:  portera@msu.edu
www.humanpathology.msu.edu

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

Message: 14
Date: Wed, 01 Feb 2006 15:35:54 -0600
From: LuAnn Anderson 
Subject: Re: [Histonet] Tissue Processor Paraffin Temps for CAP
	Inspections
To: Rene J Buesa , Amy Porter ,
	Histonet 
Message-ID: <6.2.3.4.0.20060201153334.01d663e0@ander093.email.umn.edu>
Content-Type: text/plain; charset="iso-8859-1"; format=flowed

I do remember that you are required to use a 
separate thermometer which has been calibrated 
against a thermostandard and that temps from the 
digital readouts were not to be used. If you'd 
like, I can try to find where I read that.
LuAnn



At 03:22 PM 2/1/2006, Rene J Buesa wrote:
>Amy:
>   The digital thermometer in the VIP is not 
> only very accurate but the most expeditious way of checking the
temperature.
>   Just prepare a log that include all your VIPs 
> and record the temperature once a day.
>   That is the way I did it, and never had problems with CAP inspectors.
>   Hope this will help you.
>   René J.
>
>Amy Porter  wrote:
>   If your willing to share - how are people 
> monitoring the temperature of paraffin baths on 
> tissue processors? Are you using additional 
> thermometers directly into the paraffin baths, 
> or just going by the digital read out on the 
> display for the (oven on a VIP) instrument? 
> Just curious how everyone is dealing with this 
> particular item. Thanks in advance for the many 
> responses and ideas I know I will get from asking this question!
>Amy S. Porter, HT(ASCP) QIHC
>Laboratory Supervisor
>Michigan State University
>Investigative HistoPathology Laboratory
>Department of Physiology / Division of Human Pathology
>2100 Biomedical Physical Sciences Bldg. Room #2133
>East Lansing, MI 48824-3320
>Phone: (517) 355-6475 ext 1480 / Fax: (517) 432-1368
>Email: portera@msu.edu
>www.humanpathology.msu.edu
>
>_______________________________________________
>Histonet mailing list
>Histonet@lists.utsouthwestern.edu
>http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>
>
>
>
>
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------------------------------

Message: 15
Date: Wed, 1 Feb 2006 15:03:56 -0800
From: "Goodpaster, Tracy A" 
Subject: RE: [Histonet] Active caspase 3 staining of mouse small
	intestine
To: "Yu, Jian" ,	
Message-ID:
	<8BD501B158A381409FE54DB421F82FCA02E8BA64@groucho.fhcrc.org>
Content-Type: text/plain;	charset="US-ASCII"

We use the cleaved caspase-3 antibody from Biocare with good success.
Previously, we used the one from Cell Signalling, but switched after a
side by side comparison and cost evaluation.  We steam heat formalin
fixed, paraffin embedded sections for 15 minutes in Dako's pH10 antigen
retrieval buffer.  We then use an Avidin/biotin block and serum block
(15% goat + 5% human in antibody dilutent).  We incubate the antibody
for 90 minutes and detect it with a biotinylated goat anti-rabbit IgG at
1:400, the Vector elite RTU ABC and Dako DAB plus.  We get crisp
staining on mouse, dog and human tissue.  I hope this helps. 

-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu
[mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of Yu, Jian
Sent: Wednesday, February 01, 2006 9:56 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Active caspase 3 staining of mouse small intestine

Thanks to several of you who have given me a lot of great suggestions on
how to get good cross sections of mouse small intestine   One of you
mentioned that you do Caspase 3 staining routinely with the small
intestine, sorry that I could not find your email anyone.  I have been
using the CAM1 Ab from BD, which works well for IF on frozen sections
but have a lot of background for IHC.  Unfortunately the frozen sections
do not have the greatest structures.  I would very much appreciate that
if you could share your experience with paraffin sections.

 

Thanks again!

********************************************************

Jian Yu, Ph. D.

University of Pittsburgh Cancer Institute

Hillman Cancer Center Research Pavilion

Office suite 2.26h, Laboratory 2.43

5117 Centre Avenue, Pittsburgh, PA 15213

 

Phone : 412-623-7786, (Lab) 412-623-3255

Fax:      412-623-7778

Email:   yuj2@upmc.edu

********************************************************

 

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

Message: 16
Date: Wed, 1 Feb 2006 23:34:32 -0000
From: "Jonathan Wilson" 
Subject: [Histonet] citation for negative controls
To: 
Message-ID: <00b901c62788$0e10b7d0$146fab81@COBITIS>
Content-Type: text/plain;	charset="iso-8859-1"

Hello,
I use IHC in basic research and have been digging through the archives for
information on appropriate negative controls and came across the use of
irrelavent antibodies. I would like to use this information in a paper I am
writing and was wondering if this is cited in a review or paper. I have
Harlow and Lane's book but it doesn't mention this type of control.
Thank you in advance for any help.
Sincerely,
Jon


Jonathan Wilson (PhD)
ecofisiologia CIMAR
Rua dos Bragas 289
4050-123 Porto Portugal
office 351 22 340 1809
lab 351 22 340 1834
fax 351 22 339 0608

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

Message: 17
Date: Thu, 2 Feb 2006 11:01:00 +1100
From: "Tony Henwood" 
Subject: RE: [Histonet] citation for negative controls
To: "Jonathan Wilson" ,
	
Message-ID: 
Content-Type: text/plain; charset="us-ascii"

Jonathan,
The following references might be of use:

Childs (1983) J Histochem Cytochem 31 (1A):168-176
Petrusz (1983) J Histochem Cytochem 31(1A):177-179.

Regards

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager & Senior Scientist
The Children's Hospital at Westmead,
Locked Bag 4001, Westmead, 2145, AUSTRALIA.
Tel: 612 9845 3306
Fax: 612 9845 3318




-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu
[mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of Jonathan
Wilson
Sent: Thursday, 2 February 2006 10:35 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] citation for negative controls


Hello,
I use IHC in basic research and have been digging through the archives
for information on appropriate negative controls and came across the use
of irrelavent antibodies. I would like to use this information in a
paper I am writing and was wondering if this is cited in a review or
paper. I have Harlow and Lane's book but it doesn't mention this type of
control. Thank you in advance for any help. Sincerely, Jon


Jonathan Wilson (PhD)
ecofisiologia CIMAR
Rua dos Bragas 289
4050-123 Porto Portugal
office 351 22 340 1809
lab 351 22 340 1834
fax 351 22 339 0608 _______________________________________________
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

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

Message: 18
Date: Thu, 2 Feb 2006 08:58:33 -0000 
From: Kemlo Rogerson 
Subject: RE: [Histonet] processing 
To: "'Gayle Callis'" , Kemlo Rogerson
	,
Histonet@lists.utsouthwestern.edu
Message-ID:
	

Content-Type: text/plain

My point stands though. What makes certain animal tissue more susceptible to
hardening? You eloquently explain how we solve the problem but I've never
seen the problem defined.

Surely all animal tissue is intrinsically the same? So why does it process
differently? Now I'm really rambling; why does it taste differently?

Kemlo Rogerson
Pathology Manager
Ext  3311
DD   01934 647057
Mob 07749 754194
 
 
 
-----Original Message-----
From: Gayle Callis [mailto:gcallis@montana.edu] 
Sent: Wednesday, February 01, 2006 6:35 PM
To: Kemlo Rogerson; Histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] processing 

You are not rambling.   Animal tissues, particularly rodent are very lean, 
and if one really wants to see dry animal tissue, tissues from birds of 
prey (hawks), other birds and reptiles can be among the worst. We custom 
process our rodent tissues, brain, small, etc and have shorter schedules 
for mouse brain versus hamster brain, that type of thing just to avoid 
overdehydration.

The 1 hour per station or change even with 3 X 95% and 3 x 100% never seems 
to bother human species tissues much, but with rodent tissue, it removes 
too much of the bound water.  some larger animals with bigger samples in a 
cassette seem to be ok with standard processing schedules but never with 
heat added.

A curious thing observed over a long career in histowork, I saw the 
addition of heat to processing in automated processor a curious 
thing.  When we used the older carousel (Sp?)  models i.e Technicon, heat 
couldn't be added and processing was done at RT.  True, heat may speed up 
the solvent exchange but it also can add to drying of tissues.  I have 
always wondered why this has become a standard method over the years other 
than trying to speed up processing, but always felt alternating vacuum and 
pressure to be the best way to do that along with paying attention to 
length of processing schedules.

What one needs to do is find the correct balance so that the free water in 
the tissue spaces is removed and not the bound water on protein 
molecules.  Overdehydration and adding heat to processing will exacerbate 
the removal of bound water. Consequently, heat is never added to our animal 
tissue processing steps.




  At 02:38 AM 2/1/2006, you wrote:
>Think rodent tissue can process on the 'hard' side. Wonder what makes it
>'hard'? Is it that the bonds between the proteins are 'stronger' or the
>proteins are 'nearer' together? I mean if we could figure out what made
>tissue 'hard' molecularly then we'd know which the culprit in the
processing
>was.
>
>Sorry to ramble.
>
>Kemlo Rogerson
>Pathology Manager
>Ext  3311
>DD   01934 647057
>Mob 07749 754194
>
>
>
>
>-----Original Message-----
>From: Steven Coakley [mailto:sjchtascp@yahoo.com]
>Sent: Tuesday, January 31, 2006 6:56 PM
>To: Histonet@lists.utsouthwestern.edu
>Subject: [Histonet] processing
>
>Good afternoon,
>
>   I'm working with mouse/rat muscle and trying to fine tune my processing
>schecdule.
>   My last run of muscle were very dry.  I have a copy of the NSH Animal
>Processing Manual and trying to strike a balance between those listed on
>page 5-6.  I've noticed most of the protocols do not call for heat or P/V.
>The scedule I currently use is as follows: 1-70% (hold), 1-80%, 2-95%,
>3-100%, 3-xylene all for 30 minutes each and at 38C with P/V.  3 paraffins,
>45 minutes each at 55C with P/V.  I'm fairly new to research and still
>trying to make the "mental" switch in handling the different tissue.
>
>   Thanks everyone,
>
>   Steve
>
>
>
>---------------------------------
>Bring words and photos together (easily) with
>  PhotoMail  - it's free and works with Yahoo! Mail.
>_______________________________________________
>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

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)




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

Message: 19
Date: Thu, 2 Feb 2006 05:47:20 -0600
From: "Molinari, Betsy" 
Subject: RE: [Histonet] Tissue Processor Paraffin Temps for CAP
	Inspections
To: 
Message-ID: 
Content-Type: text/plain;	charset="us-ascii"

Once a month I put a calibrated thermometer in one of the paraffin
reservoirs.

Betsy Molinari HT (ASCP)
Texas Heart Institute
Cardiovascular Pathology
6770 Bertner Ave.
Houston,TX 77030
832-355-6524
832-355-6812 (fax)


-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu
[mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of Amy
Porter
Sent: Wednesday, February 01, 2006 3:13 PM
To: Histonet
Subject: [Histonet] Tissue Processor Paraffin Temps for CAP Inspections

If your willing to share - how are people monitoring the temperature of
paraffin baths on tissue processors?  Are you using additional
thermometers directly into the paraffin baths, or just going by the
digital read out on the display for the (oven on a VIP) instrument?
Just curious how everyone is dealing with this particular item.  Thanks
in advance for the many responses and ideas I know I will get from
asking this question!
Amy S. Porter, HT(ASCP) QIHC
Laboratory Supervisor
Michigan State University
Investigative HistoPathology Laboratory 
Department of Physiology / Division of Human Pathology
2100 Biomedical Physical Sciences Bldg. Room #2133
East Lansing, MI  48824-3320
Phone:  (517) 355-6475 ext 1480 / Fax:  (517) 432-1368
Email:  portera@msu.edu
www.humanpathology.msu.edu

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



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

Message: 20
Date: Thu, 2 Feb 2006 13:29:53 +0000
From: Susan.Ferrigon@sanofi-aventis.com
Subject: [Histonet] Kuppfer cells
To: histonet@lists.utsouthwestern.edu
Message-ID:
	
Content-Type: text/plain; charset=ISO-8859-1





Hi

Can anyone suggest a marker for Kuppfer cells??

Thanks

Susan
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retaining a copy.
------------------------------




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

Message: 21
Date: Thu, 2 Feb 2006 09:43:58 -0500
From: "Adams, Nancy" 
Subject: [Histonet] microwave transparent containers and equipment
To: 
Message-ID:
	
<17A1862099540D458C8FE9380C2BC461C0932E@fh2xmail.fhdomain1.capecodhealth.org
>
	
Content-Type: text/plain

 

            Good morning

 

Does the new CAP question ANP.28860 require documentation that the
containers and equipment are,  indeed, microwave transparent?  

 

Somehow I think just saying yes isn't going to be enough:-)

 

Thanks for your thoughts.

 

Nancy Rutledge

Falmouth Hospital



************************************************************
This email and any files transmitted with it are confidential. And intended
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------------------------------

Message: 22
Date: Thu, 2 Feb 2006 07:28:05 -0800 (PST)
From: GT Hebert 
Subject: Re: [Histonet] Kuppfer cells
To: Susan.Ferrigon@sanofi-aventis.com
Cc: histonet@lists.utsouthwestern.edu
Message-ID: <20060202152805.96087.qmail@web31704.mail.mud.yahoo.com>
Content-Type: text/plain; charset=iso-8859-1

Susan,
   
  I've used F4/80.  Though I was trying to stain macrophages, I used the
Kupffer cells in the liver as a positive control to show that the antibody
was working and they stained perfectly.
   
  Gustave Hebert
  Scientist II
  Wyeth Research 
  Cambridge MA
   
  
Susan.Ferrigon@sanofi-aventis.com wrote:
  



Hi

Can anyone suggest a marker for Kuppfer cells??

Thanks

Susan
------------------------------
Le présent message ainsi que ses éventuelles pièces jointes est
exclusivement destiné au(x) destinataire(s), personnes physiques ou
morales, qu'il désigne.
Il constitue de ce fait une correspondance à caractère privé et peut
contenir des informations confidentielles.
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retaining a copy.
------------------------------


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and used cars.

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

Message: 23
Date: Thu, 2 Feb 2006 07:44:20 -0800 (PST)
From: Rene J Buesa 
Subject: Re: [Histonet] microwave transparent containers and equipment
To: "Adams, Nancy" ,
	histonet@lists.utsouthwestern.edu
Message-ID: <20060202154420.27452.qmail@web61225.mail.yahoo.com>
Content-Type: text/plain; charset=iso-8859-1

Nancy:
  You could show them a list with the microwaves penetration (cm) of
different substances.
  For example: 
  polyethyl methacrylate = 300 cm (= 3 m)
  Teflon = 9,000 cm (= 90 m)
  Paraffin wax = 15,000 (=150 m)
  Styrofoam= 40,000 cm (=400 m) 
   
  Showing them the materials your containers are made of I think that they
will
  accept that they are indeed transparent.
  Hope this will help.
  René J.

"Adams, Nancy"  wrote:
  

Good morning



Does the new CAP question ANP.28860 require documentation that the
containers and equipment are, indeed, microwave transparent? 



Somehow I think just saying yes isn't going to be enough:-)



Thanks for your thoughts.



Nancy Rutledge

Falmouth Hospital



************************************************************
This email and any files transmitted with it are confidential. And intended
solely for the use of the individual or entity to whom they are addressed.
If you have received this email in error please contact the system
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Administrator@CapeCodHealth.org
************************************************************

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Message: 24
Date: Thu, 2 Feb 2006 17:09:19 +0100
From: "Bruijntjes, J.P." 
Subject: FW: [Histonet] Tissue Processor Paraffin Temps for CAP
	Inspections
To: 
Message-ID:
	<3B070848E7C2204F9DEB8BCFD767728004A69837@ntexch1.voeding.tno.nl>
Content-Type: text/plain;	charset="us-ascii"

Hi Amy

We use an electronic thermometer (testo 915-1) which is calibrated once
or twice a year. 

Joost Bruijntjes
TNO Quality of Life
Toxicology and Applied Pharmacology
Zeist
Holland

-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu
[mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of
Molinari, Betsy
Sent: donderdag 2 februari 2006 12:47
To: histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] Tissue Processor Paraffin Temps for CAP
Inspections

Once a month I put a calibrated thermometer in one of the paraffin
reservoirs.

Betsy Molinari HT (ASCP)
Texas Heart Institute
Cardiovascular Pathology
6770 Bertner Ave.
Houston,TX 77030
832-355-6524
832-355-6812 (fax)


-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu
[mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of Amy
Porter
Sent: Wednesday, February 01, 2006 3:13 PM
To: Histonet
Subject: [Histonet] Tissue Processor Paraffin Temps for CAP Inspections

If your willing to share - how are people monitoring the temperature of
paraffin baths on tissue processors?  Are you using additional
thermometers directly into the paraffin baths, or just going by the
digital read out on the display for the (oven on a VIP) instrument?
Just curious how everyone is dealing with this particular item.  Thanks
in advance for the many responses and ideas I know I will get from
asking this question!
Amy S. Porter, HT(ASCP) QIHC
Laboratory Supervisor
Michigan State University
Investigative HistoPathology Laboratory 
Department of Physiology / Division of Human Pathology
2100 Biomedical Physical Sciences Bldg. Room #2133
East Lansing, MI  48824-3320
Phone:  (517) 355-6475 ext 1480 / Fax:  (517) 432-1368
Email:  portera@msu.edu
www.humanpathology.msu.edu

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

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

Message: 25
Date: Thu, 02 Feb 2006 10:02:18 -0700
From: Gayle Callis 
Subject: Re: [Histonet] citation for negative controls
To: "Jonathan Wilson" ,
	Histonet@lists.utsouthwestern.edu
Message-ID:
	<6.0.0.22.1.20060202095040.01b3ce58@gemini.msu.montana.edu>
Content-Type: text/plain; charset="us-ascii"; format=flowed

Appropriate negative controls are discussed by Boenisch, in DAKO 3rd 
Edition Handbook Immunochemical Staining methods, and found on the DAKO 
website as a free pdf download.  It can be done and clinical laboratories 
do use irrelevant antibodies at times also.  Jules Elias books also discuss 
this, Immunohistopathology, a pracatical approach to diagnostics, ASCP 
press. There is a second edition although may not be available to you.

If you are working with mouse, this can be a difficult thing to find at 
times so that you get NO staining with an irrelevant 
antibody.  Consequently we use what is most cited in the literature, 
isotype matched immunoglobulin i.e. IgG controls depending on species and 
immunoglobulin.  They come pure, biotinylated, conjugated to many things 
including Alexa dyes.  OR you can buy whole IgG's from Jackson (whole rat 
IgG contains all the isotypes, and from many different species.)

One that happened some time ago, we used rat serum as a  control and the 
reviewers balked big time.  We had to REPEAT what was a very difficult, 
tedious immunogold staining project using isotype matched controls.   Since 
that time, we NEVER use normal serums nor an irrelevant antibody for a 
negative control.   Having to repeat work is not fun!



  At 04:34 PM 2/1/2006, you wrote:
>Hello,
>I use IHC in basic research and have been digging through the archives for 
>information on appropriate negative controls and came across the use of 
>irrelavent antibodies. I would like to use this information in a paper I 
>am writing and was wondering if this is cited in a review or paper. I have 
>Harlow and Lane's book but it doesn't mention this type of control.
>Thank you in advance for any help.
>Sincerely,
>Jon
>
>
>Jonathan Wilson (PhD)
>ecofisiologia CIMAR
>Rua dos Bragas 289
>4050-123 Porto Portugal
>office 351 22 340 1809
>lab 351 22 340 1834
>fax 351 22 339 0608
>_______________________________________________
>Histonet mailing list
>Histonet@lists.utsouthwestern.edu
>http://lists.utsouthwestern.edu/mailman/listinfo/histonet

Gayle Callis HTL, HT, MT(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717




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

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