[Histonet] Breast processing
Martin, Gary
gmartin <@t> marshallmedical.org
Mon Oct 8 13:52:26 CDT 2012
We have someone come in over the weekend and embed the tissue. If we go over the time protocol for some reason, which is not often, we will evaluate the specimen by FISH. Once you take the bitter pill of someone coming over the weekend, it works very well.
Gary
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of histonet-request <@t> lists.utsouthwestern.edu
Sent: Monday, October 08, 2012 10:02 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: Histonet Digest, Vol 107, Issue 10
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Today's Topics:
1. RE: Breast processing times (Weems, Joyce K.)
2. Brain - Question and advise (Histo Histo)
3. back online, please feel free to check it out (M. Kap)
4. Flourescent mounting medium- PLEASE HELP!!! (Candice Smoots)
5. RE: Flourescent mounting medium- PLEASE HELP!!! (Jonathan Cremer)
6. Re: Brain - Question and advise (Rene J Buesa)
7. RE: Brain - Question and advise (McMahon, Loralee A)
8. question about decalicification end-point checks
(Thurby, Christina)
9. Re: Brain - Question and advise (Rene J Buesa)
10. Re: Flourescent mounting medium- PLEASE HELP!!! (Rene J Buesa)
11. SOX-10 (Houston, Ronald)
12. RE: SOX-10 (Della Speranza, Vinnie)
13. Re: question about decalicification end-point checks
(Rene J Buesa)
----------------------------------------------------------------------
Message: 1
Date: Mon, 8 Oct 2012 00:33:13 +0000
From: "Weems, Joyce K." <Joyce.Weems <@t> emoryhealthcare.org>
Subject: [Histonet] RE: Breast processing times
To: "Rathborne, Toni" <trathborne <@t> somerset-healthcare.com>, Diana
McCaig <dmccaig <@t> ckha.on.ca>, "histonet <@t> lists.utsouthwestern.edu"
<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<E3A4EBD57A691646BCCED4AA5911A030667AF67A <@t> e14mbx12n.Enterprise.emory.net>
Content-Type: text/plain; charset="iso-8859-1"
We have someone from the clinical lab drain the processor, remove the blocks and set them aside for us to complete on Monday morning. Has worked perfectly for us. j
________________________________________
From: histonet-bounces <@t> lists.utsouthwestern.edu [histonet-bounces <@t> lists.utsouthwestern.edu] on behalf of Rathborne, Toni [trathborne <@t> somerset-healthcare.com]
Sent: Friday, October 05, 2012 11:18 AM
To: Diana McCaig; histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] RE: Breast processing times
We have someone come in on Sunday, and work with one less person on Monday.
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Diana McCaig
Sent: Friday, October 05, 2012 11:15 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Breast processing times
Can you please give me your routine for processing breast cases over the weekend. Do you extend the time in alcohol to achieve the 48 hour maximum, call someone in over the weekend, or just indicate on the patient report that the fixation extended beyond the 48 hours limit for HER 2, 72 hours for ER and PR. Is the ischemic time recorded within the lab as well as on the patient report?
Thanks
Diana
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Message: 2
Date: Sun, 7 Oct 2012 22:12:28 -0700
From: Histo Histo <feulgenreaction <@t> gmail.com>
Subject: [Histonet] Brain - Question and advise
To: histonet <@t> lists.utsouthwestern.edu
Message-ID:
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Hi all,
Our company is planning to start receiving formalin fixed,human brain tumor
samples from their clients. I have not personally worked with brain in my
career and was hoping to get some pointers from my fellow histoneters.
I was hoping to get some advise on the following:
- Any special precautions needed to be taken when handling these samples
(prions, ect)?
- Section thickness
- Staining setup for H&E
- Common artifacts
- Any other pointers you may have.
Thank you in advance!
- F
------------------------------
Message: 3
Date: Mon, 8 Oct 2012 08:53:44 +0000
From: "M. Kap" <m.kap.1 <@t> erasmusmc.nl>
Subject: [Histonet] back online, please feel free to check it out
To: "histonet <@t> lists.utsouthwestern.edu"
<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<7CD0BD0DBF66F94F96E9A75A96D7F8EFE26E22 <@t> EXCH-RX02.erasmusmc.nl>
Content-Type: text/plain; charset="us-ascii"
Dear Histonetters,
Some of you visited the Facebook page to find some nice pictures instead of the CQPath site, which was disappointingly down. After a server crash this weekend, we are back online! Once again I want to kindly invite you to take a look at our IHC QC/QA/QI program.
Please take a few clicks to visit http://www.cqpath.com/index.htm?language=en and please leave your comments at our Facebook page https://www.facebook.com/CQPath
This is all free and might encourage you to contact us for more info.
We hope you like the program. Please let us know anything at all, all feedback is important and useful! (Yess, even when you think it s#cks)
Best regards,
Marcel
------------------------------
Message: 4
Date: Mon, 8 Oct 2012 06:42:52 -0700 (PDT)
From: Candice Smoots <candice_camille <@t> yahoo.com>
Subject: [Histonet] Flourescent mounting medium- PLEASE HELP!!!
To: Histonet <histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<1349703772.7292.YahooMailNeo <@t> web165005.mail.bf1.yahoo.com>
Content-Type: text/plain; charset=iso-8859-1
Hi Histonet
In our lab, we are trying to mount our flourescent stained sections with polyvinyl alcohol mounting medium with DABCO. WE are unsure if it is to be mounted while the sections are wet or do we let me air dry.
Another question is... How to actually use it. It looks to be a little solidified. Kind of like jello. Is this normal? do I dilute it? Please Please Help!
I remain yours truely,
Candice Camille
------------------------------
Message: 5
Date: Mon, 8 Oct 2012 14:20:07 +0000
From: Jonathan Cremer <Jonathan.Cremer <@t> med.kuleuven.be>
Subject: RE: [Histonet] Flourescent mounting medium- PLEASE HELP!!!
To: Candice Smoots <candice_camille <@t> yahoo.com>, Histonet
<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<C35C2709C22FC2458D6A918FACC338A30FA6C926 <@t> ICTS-S-MBX5.luna.kuleuven.be>
Content-Type: text/plain; charset="us-ascii"
I have no experience with this particular mounting medium, but from what I gather it is like any other aqueous mounting medium. You should blot your slides to remove as much water as possible, without letting your sections dry. Then apply a small amount of mounting medium to the slide or coverslip (depending on preference) and mount.
Apparently it solidifies at the edge, so no need to seal with nail polish.
If your bottle has solidified to the point where it is a gel instead of a viscous liquid, you can toss it. (unless the datasheet specifies otherwise...)
Jonathan
________________________________________
Van: histonet-bounces <@t> lists.utsouthwestern.edu [histonet-bounces <@t> lists.utsouthwestern.edu] namens Candice Smoots [candice_camille <@t> yahoo.com]
Verzonden: maandag 8 oktober 2012 15:42
To: Histonet
Onderwerp: [Histonet] Flourescent mounting medium- PLEASE HELP!!!
Hi Histonet
In our lab, we are trying to mount our flourescent stained sections with polyvinyl alcohol mounting medium with DABCO. WE are unsure if it is to be mounted while the sections are wet or do we let me air dry.
Another question is... How to actually use it. It looks to be a little solidified. Kind of like jello. Is this normal? do I dilute it? Please Please Help!
I remain yours truely,
Candice Camille
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------------------------------
Message: 6
Date: Mon, 8 Oct 2012 07:51:55 -0700 (PDT)
From: Rene J Buesa <rjbuesa <@t> yahoo.com>
Subject: Re: [Histonet] Brain - Question and advise
To: Histo Histo <feulgenreaction <@t> gmail.com>,
"histonet <@t> lists.utsouthwestern.edu"
<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<1349707915.7093.YahooMailNeo <@t> web163105.mail.bf1.yahoo.com>
Content-Type: text/plain; charset=iso-8859-1
If you will receive brain tumors, you should not worry about prions. On the other hand, you should ask the referring physicians the nature of the lesion to be on the "safe" side.
The special procedural "tips" for brains can be summed up as:
1- make sure they are fixed correctly (at least 48 hours fixation) and the thickness of the slices should be about 2mm
2- dehydration must be perfect and gradual
3- increase the "infiltration" step at twice the time of your standard protocol
4- section thickness should be 5 µm and if you are going to do any silver stains, increase it to 8 µm so you can demonstrate the neuronal web
5- the H&E setup can be the standard you use
6- cool the blocks before sectioning
René J.
________________________________
From: Histo Histo <feulgenreaction <@t> gmail.com>
To: histonet <@t> lists.utsouthwestern.edu
Sent: Monday, October 8, 2012 1:12 AM
Subject: [Histonet] Brain - Question and advise
Hi all,
Our company is planning to start receiving formalin fixed,human brain tumor
samples from their clients. I have not personally worked with brain in my
career and was hoping to get some pointers from my fellow histoneters.
I was hoping to get some advise on the following:
- Any special precautions needed to be taken when handling these samples
(prions, ect)?
- Section thickness
- Staining setup for H&E
- Common artifacts
- Any other pointers you may have.
Thank you in advance!
- F
_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
------------------------------
Message: 7
Date: Mon, 8 Oct 2012 11:18:39 -0400
From: "McMahon, Loralee A" <Loralee_Mcmahon <@t> URMC.Rochester.edu>
Subject: RE: [Histonet] Brain - Question and advise
To: Rene J Buesa <rjbuesa <@t> yahoo.com>, Histo Histo
<feulgenreaction <@t> gmail.com>, "histonet <@t> lists.utsouthwestern.edu"
<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<EC3287D73321A14799BB96082DE57B476BCD4AB4 <@t> URMCMS2.urmc-sh.rochester.edu>
Content-Type: text/plain; charset="iso-8859-1"
Curious as to why you wouldn't "worry about prions" ?
Loralee McMahon, HTL (ASCP)
Immunohistochemistry Supervisor
Strong Memorial Hospital
Department of Surgical Pathology
(585) 275-7210
________________________________________
From: histonet-bounces <@t> lists.utsouthwestern.edu [histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Rene J Buesa [rjbuesa <@t> yahoo.com]
Sent: Monday, October 08, 2012 10:51 AM
To: Histo Histo; histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] Brain - Question and advise
If you will receive brain tumors, you should not worry about prions. On the other hand, you should ask the referring physicians the nature of the lesion to be on the "safe" side.
The special procedural "tips" for brains can be summed up as:
1- make sure they are fixed correctly (at least 48 hours fixation) and the thickness of the slices should be about 2mm
2- dehydration must be perfect and gradual
3- increase the "infiltration" step at twice the time of your standard protocol
4- section thickness should be 5 µm and if you are going to do any silver stains, increase it to 8 µm so you can demonstrate the neuronal web
5- the H&E setup can be the standard you use
6- cool the blocks before sectioning
René J.
________________________________
From: Histo Histo <feulgenreaction <@t> gmail.com>
To: histonet <@t> lists.utsouthwestern.edu
Sent: Monday, October 8, 2012 1:12 AM
Subject: [Histonet] Brain - Question and advise
Hi all,
Our company is planning to start receiving formalin fixed,human brain tumor
samples from their clients. I have not personally worked with brain in my
career and was hoping to get some pointers from my fellow histoneters.
I was hoping to get some advise on the following:
- Any special precautions needed to be taken when handling these samples
(prions, ect)?
- Section thickness
- Staining setup for H&E
- Common artifacts
- Any other pointers you may have.
Thank you in advance!
- F
_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
------------------------------
Message: 8
Date: Mon, 8 Oct 2012 11:35:48 -0400
From: "Thurby, Christina" <christina.thurby <@t> bms.com>
Subject: [Histonet] question about decalicification end-point checks
To: "histonet <@t> lists.utsouthwestern.edu"
<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<E9C77491626EBF41BD449EA1098B040608719D02C6 <@t> ushpwbmsmmp007.one.ads.bms.com>
Content-Type: text/plain; charset="us-ascii"
Hi,
I am looking for help with decalcification end-point checks. We use a 5% Formic acid decal (homemade - 1 L 88% Formic acid, 1 L 37% Formaldehyde and 8 L of distilled water). We do an endpoint check using 5% ammonium oxylate. Recently, we decaled canine femurs for a total of 13 days and endpoint check was performed on days 12 and 13. The femurs were fixed in 10% NBF and then sectioned with a band saw prior to decalcification. The decal endpoint tested out on both days 12 and 13. Our problem is at microtomy, the center of the bones do not appear to be completely decaled and we can not get a good section. We have tried surface decaling the blocks with no luck. Any thoughts? Is there a commercially available decal endpoint check that others have used for acid decals?
I have just finished reading Mr. Skinner & Ms. Schray's article "Decalicification of Mouse Bone/Soft Tissue Composit Specimens" from mikro-graf publishing, Volume 41, Issue 01 - this article does give some good information about paraffin embedding (which may be part of our issue on these larger bone specimens). Perhaps we may look into the 50/50 embedding medium at a higher temp for large animal bone specimens like this in the future which seem to yield good results in Mr. Skinner's laboratory.
Robert or Carrie - if one of you are available could you give me a call or e-mail to discuss? Thanks so much!! Any feedback will be greatly appreciated!
Kristie Thurby
Bristol Myers Squibb
812-307-2093
________________________________
This message (including any attachments) may contain confidential, proprietary, privileged and/or private information. The information is intended to be for the use of the individual or entity designated above. If you are not the intended recipient of this message, please notify the sender immediately, and delete the message and any attachments. Any disclosure, reproduction, distribution or other use of this message or any attachments by an individual or entity other than the intended recipient is prohibited.
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Message: 9
Date: Mon, 8 Oct 2012 09:03:22 -0700 (PDT)
From: Rene J Buesa <rjbuesa <@t> yahoo.com>
Subject: Re: [Histonet] Brain - Question and advise
To: "McMahon, Loralee A" <Loralee_Mcmahon <@t> URMC.Rochester.edu>, Histo
Histo <feulgenreaction <@t> gmail.com>, "histonet <@t> lists.utsouthwestern.edu"
<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<1349712202.23394.YahooMailNeo <@t> web163101.mail.bf1.yahoo.com>
Content-Type: text/plain; charset=iso-8859-1
I do not worry about prions IF the tumor is perfectly classified as a metastatic neoplasm or any nervous tissue tumor.
"Prions", as you know, correspond to a pathology with very well defined behaviors consistent with the human form of "mad cow disease" (KJ disease).
That is why I recommended to get a diagnosis from the referring physician.
René J.
________________________________
From: "McMahon, Loralee A" <Loralee_Mcmahon <@t> URMC.Rochester.edu>
To: Rene J Buesa <rjbuesa <@t> yahoo.com>; Histo Histo <feulgenreaction <@t> gmail.com>; "histonet <@t> lists.utsouthwestern.edu" <histonet <@t> lists.utsouthwestern.edu>
Sent: Monday, October 8, 2012 11:18 AM
Subject: RE: [Histonet] Brain - Question and advise
Curious as to why you wouldn't "worry about prions" ?
Loralee McMahon, HTL (ASCP)
Immunohistochemistry Supervisor
Strong Memorial Hospital
Department of Surgical Pathology
(585) 275-7210
________________________________________
From: histonet-bounces <@t> lists.utsouthwestern.edu [histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Rene J Buesa [rjbuesa <@t> yahoo.com]
Sent: Monday, October 08, 2012 10:51 AM
To: Histo Histo; histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] Brain - Question and advise
If you will receive brain tumors, you should not worry about prions. On the other hand, you should ask the referring physicians the nature of the lesion to be on the "safe" side.
The special procedural "tips" for brains can be summed up as:
1- make sure they are fixed correctly (at least 48 hours fixation) and the thickness of the slices should be about 2mm
2- dehydration must be perfect and gradual
3- increase the "infiltration" step at twice the time of your standard protocol
4- section thickness should be 5 µm and if you are going to do any silver stains, increase it to 8 µm so you can demonstrate the neuronal web
5- the H&E setup can be the standard you use
6- cool the blocks before sectioning
René J.
________________________________
From: Histo Histo <feulgenreaction <@t> gmail.com>
To: histonet <@t> lists.utsouthwestern.edu
Sent: Monday, October 8, 2012 1:12 AM
Subject: [Histonet] Brain - Question and advise
Hi all,
Our company is planning to start receiving formalin fixed,human brain tumor
samples from their clients. I have not personally worked with brain in my
career and was hoping to get some pointers from my fellow histoneters.
I was hoping to get some advise on the following:
- Any special precautions needed to be taken when handling these samples
(prions, ect)?
- Section thickness
- Staining setup for H&E
- Common artifacts
- Any other pointers you may have.
Thank you in advance!
- F
_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
------------------------------
Message: 10
Date: Mon, 8 Oct 2012 09:06:24 -0700 (PDT)
From: Rene J Buesa <rjbuesa <@t> yahoo.com>
Subject: Re: [Histonet] Flourescent mounting medium- PLEASE HELP!!!
To: Candice Smoots <candice_camille <@t> yahoo.com>, Histonet
<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<1349712384.64144.YahooMailNeo <@t> web163103.mail.bf1.yahoo.com>
Content-Type: text/plain; charset=iso-8859-1
The sections should be wet (usually in PBS).
If your mounting medium is a "little solidified" you should dilute it with the same diluent specified in the bottle or the MSDS
René J.
________________________________
From: Candice Smoots <candice_camille <@t> yahoo.com>
To: Histonet <histonet <@t> lists.utsouthwestern.edu>
Sent: Monday, October 8, 2012 9:42 AM
Subject: [Histonet] Flourescent mounting medium- PLEASE HELP!!!
Hi Histonet
In our lab, we are trying to mount our flourescent stained sections with polyvinyl alcohol mounting medium with DABCO. WE are unsure if it is to be mounted while the sections are wet or do we let me air dry.
Another question is... How to actually use it. It looks to be a little solidified. Kind of like jello. Is this normal? do I dilute it? Please Please Help!
I remain yours truely,
Candice Camille
_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
------------------------------
Message: 11
Date: Mon, 8 Oct 2012 16:24:22 +0000
From: "Houston, Ronald" <Ronald.Houston <@t> nationwidechildrens.org>
Subject: [Histonet] SOX-10
To: "histonet <@t> lists.utsouthwestern.edu"
<histonet <@t> lists.utsouthwestern.edu>, "ihcrg <@t> googlegroups.com"
<ihcrg <@t> googlegroups.com>
Message-ID:
<E5D0CD2352E46545A0C0EBE308CCCE5301BA9AB3 <@t> L1PERDWXMB02.childrensroot.net>
Content-Type: text/plain; charset="us-ascii"
Is anyone using SOX 10 in the diagnosis of soft tissue tumors? If so, can you please recommend a good reliable source for the antibody?
Thanks
Ronnie Houston, MS HT(ASCP)QIHC
Anatomic Pathology Manager
ChildLab, a Division of Nationwide Children's Hospital
www.childlab.com
700 Children's Drive
Columbus, OH 43205
(P) 614-722-5450
(F) 614-722-2899
ronald.houston <@t> nationwidechildrens.org<mailto:ronald.houston <@t> nationwidechildrens.org>
www.NationwideChildrens.org<http://www.nationwidechildrens.org/>
"One person with passion is better than forty people merely interested."
~ E.M. Forster
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------------------------------
Message: 12
Date: Mon, 8 Oct 2012 12:46:51 -0400
From: "Della Speranza, Vinnie" <dellav <@t> musc.edu>
Subject: [Histonet] RE: SOX-10
To: "'Houston, Ronald'" <Ronald.Houston <@t> nationwidechildrens.org>,
"histonet <@t> lists.utsouthwestern.edu"
<histonet <@t> lists.utsouthwestern.edu>, "ihcrg <@t> googlegroups.com"
<ihcrg <@t> googlegroups.com>
Message-ID:
<E58D1CD977E29C46A167806513B04577C3CEB32A48 <@t> EVS5.clinlan.local>
Content-Type: text/plain; charset="us-ascii"
Ronnie,
I've not started using this marker just yet (I've pre-ordered it for mid Oct availability), but I just received a product sheet for Cell Marque's polyclonal rabbit ab which is available in concentrate or RTU
If this interests you I can scan and email it to you (don't want to spam your inbox without your permission)
Vinnie Della Speranza
Manager for Anatomic Pathology Services
Medical University of South Carolina
165 Ashley Avenue MSC 908
Charleston, South Carolina 29425
tel. 843-792-6353
fax. 843-792-8974
CONFIDENTIALITY NOTICE
This message is intended only for the use of the individual or entity to which it is addressed and may contain information that is privileged, confidential and exempt from disclosure under applicable law. If the reader of this message is not the intended recipient, you are hereby notified that any dissemination, distribution, or copying of this communication is strictly prohibited by law. If you have received this communication in error, please notify me and the MUSC Compliance Office immediately at (843) 792-4037 or 1-800-296-0269. Thank you
________________________________
From: ihcrg <@t> googlegroups.com [mailto:ihcrg <@t> googlegroups.com] On Behalf Of Houston, Ronald
Sent: Monday, October 08, 2012 12:24 PM
To: histonet <@t> lists.utsouthwestern.edu; ihcrg <@t> googlegroups.com
Subject: [IHCRG] SOX-10
Is anyone using SOX 10 in the diagnosis of soft tissue tumors? If so, can you please recommend a good reliable source for the antibody?
Thanks
Ronnie Houston, MS HT(ASCP)QIHC
Anatomic Pathology Manager
ChildLab, a Division of Nationwide Children's Hospital
www.childlab.com
700 Children's Drive
Columbus, OH 43205
(P) 614-722-5450
(F) 614-722-2899
ronald.houston <@t> nationwidechildrens.org<mailto:ronald.houston <@t> nationwidechildrens.org>
www.NationwideChildrens.org<http://www.nationwidechildrens.org/>
"One person with passion is better than forty people merely interested."
~ E.M. Forster
________________________________
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------------------------------
Message: 13
Date: Mon, 8 Oct 2012 09:49:00 -0700 (PDT)
From: Rene J Buesa <rjbuesa <@t> yahoo.com>
Subject: Re: [Histonet] question about decalicification end-point
checks
To: "Thurby, Christina" <christina.thurby <@t> bms.com>,
"histonet <@t> lists.utsouthwestern.edu"
<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<1349714940.13779.YahooMailNeo <@t> web163105.mail.bf1.yahoo.com>
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What you describe (NBF fixation → band saw slicing → decal with formic acid → end point with 5% ammonium oxalate) seems a good protocol BUT you have to pay special attention to:
1- Did you fix the femurs "in toto"? If you did that, fixation is extremely difficult. You first should select a "general area" of interest and isolate that area with the band saw in a way that the NBF can penetrate the bone via the medullar area, otherwise fixation will be incomplete and start a "chain of irreversible problems".
2- assuming that the fixation of the whole bone was good, how thick the band saw slices to be decalcified were? They should be 5 mm thick at the most.
3- if the 2 previous issues were not completed that way you may have a (+) test for "complete decalcification" that may not reflect the reality, but lets assume the bone slices were decalcified correctly then
4- infiltration has to be extended at twice the time of your standard protocol and with a paraffin wax of at least 63ºC of melting point or ideally higher. You may not have that paraffin wax in your lab, but this is a special subject that requires special reagents if you want to arrive to a successful final section.
5- the rest will be microtomy related preferably using steel "permanent" knives, no disposable blades, with the adequate cutting angle and, ideally, with a sliding microtome
As you can see there are many variables and even under the best circumstances, bone microtomy is very difficult.
I never recommend "after standard protocol solutions" like "treating the exposed cutting surface" with acid solutions. These are desperate attempts to compensate for procedural mistakes that will never render ideal sections.
René J.
________________________________
From: "Thurby, Christina" <christina.thurby <@t> bms.com>
To: "histonet <@t> lists.utsouthwestern.edu" <histonet <@t> lists.utsouthwestern.edu>
Sent: Monday, October 8, 2012 11:35 AM
Subject: [Histonet] question about decalicification end-point checks
Hi,
I am looking for help with decalcification end-point checks. We use a 5% Formic acid decal (homemade - 1 L 88% Formic acid, 1 L 37% Formaldehyde and 8 L of distilled water). We do an endpoint check using 5% ammonium oxylate. Recently, we decaled canine femurs for a total of 13 days and endpoint check was performed on days 12 and 13. The femurs were fixed in 10% NBF and then sectioned with a band saw prior to decalcification. The decal endpoint tested out on both days 12 and 13. Our problem is at microtomy, the center of the bones do not appear to be completely decaled and we can not get a good section. We have tried surface decaling the blocks with no luck. Any thoughts? Is there a commercially available decal endpoint check that others have used for acid decals?
I have just finished reading Mr. Skinner & Ms. Schray's article "Decalicification of Mouse Bone/Soft Tissue Composit Specimens" from mikro-graf publishing, Volume 41, Issue 01 - this article does give some good information about paraffin embedding (which may be part of our issue on these larger bone specimens). Perhaps we may look into the 50/50 embedding medium at a higher temp for large animal bone specimens like this in the future which seem to yield good results in Mr. Skinner's laboratory.
Robert or Carrie - if one of you are available could you give me a call or e-mail to discuss? Thanks so much!! Any feedback will be greatly appreciated!
Kristie Thurby
Bristol Myers Squibb
812-307-2093
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End of Histonet Digest, Vol 107, Issue 10
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