[Histonet] cryostats
Molinari, Betsy
BMolinari <@t> heart.thi.tmc.edu
Thu Jun 2 06:00:29 CDT 2005
I have the Leica and it has worked very well for me.
Betsy Molinari HT(ASCP)
Texas heart Institute
Cardiovascular Pathology
Houston,TX 77030
832-355-6524
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Clarke, Mary
Sent: Wednesday, June 01, 2005 1:54 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] cryostats
We are in the process of purchasing a new cryostat. Has anyone used the Richard-Allan 525 or the Leica CM 1850? We are looking at both and wonder how people like or dislike them? Thanks in advance for your help.
Terri Clarke
Histology Supervisor
All Saints Laboratory
3801 Spring Street
Racine, Wi 53405
mclarke <@t> allsaintshealthcare.org
262-687-6609
-----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: Wednesday, June 01, 2005 12:01 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: Histonet Digest, Vol 19, Issue 1
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Today's Topics:
1. Speaking of Gram's... (Breeden, Sara)
2. Freezing muscle biopsies (Osborn, Sharon)
3. Enzyme Staining and Billing for Controls (Mitchell, Guy)
4. RE: Number of Blocks Submitted by PA (Charles.Embrey)
5. Aquaporin immunohistochemistry (Anthony Reilly)
6. Ki67. (christian <@t> eisbo.dk)
7. Ki67. (christian <@t> eisbo.dk)
8. RE: Ki67. (Bruijntjes, J.P.)
9. visualization of muscle damage with light microscopy ?
(Vincent Martin)
10. Histology of Frozen Tissue (shawnster73 <@t> aol.com)
11. BK Virus (Bobbie Boyce)
12. HPV I and II Control Blocks (WAYNE HOLLAND)
13. Re: mouse endothelial cells in brain (Corazon D. Bucana)
14. staining with Solvent Blue 38 (Donin, Nick (NIH/NCI))
15. RE: mouse endothelial cells in brain (Luis Chiriboga)
16. Storage of Frozen Section slides (CHRISTIE GOWAN)
17. Re: Will brain tissue shrink even more if
storedin70%alcoholprior to processing? (Vinnie Della Speranza)
18. RE: mouse endothelial cells in brain (Connolly, Brett M)
19. SOLVENT BLUE 38 ON PARAFFIN EMBEDDED MOUSE BRAIN (Tim Wheelock)
20. filtering hematoxylin (Diana McCaig)
21. RE: filtering hematoxylin (Bartlett, Jeanine)
----------------------------------------------------------------------
Message: 1
Date: Tue, 31 May 2005 12:33:01 -0600
From: "Breeden, Sara" <sbreeden <@t> nmda.nmsu.edu>
Subject: [Histonet] Speaking of Gram's...
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<F16987B849BBD649AA3CD478B07F81F7105206 <@t> nmdamail.nmda.ad.nmsu.edu>
Content-Type: text/plain; charset="us-ascii"
I'm having trouble with my Gram's in that my positive bacteria are
losing color. So, I'm wondering what the exact differentiation time
should be for the step after the Gram's Iodine (I'm using the Sigma
kit)? The instructions are not precise, so I'm thinkin' maybe I'm over
differentiating or under-Safranin-ing or aliens (this is the State
that's home to ROSWELL...) have taken over my kit. Anyone have anything
a bit more specific? I'd appreciate any help...
Sara A. Breeden, HT(ASCP)
New Mexico Department of Agriculture
Veterinary Diagnostic Services
POBox 4700
Albuquerque, NM 87196
(505)841-2576
(505)841-2518 FAX
------------------------------
Message: 2
Date: Tue, 31 May 2005 15:41:02 -0400
From: "Osborn, Sharon" <sharon.osborn <@t> dnax.org>
Subject: [Histonet] Freezing muscle biopsies
To: "'histonet <@t> lists.utsouthwestern.edu'"
<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<29B25753F6B1D51196110002A589D44402397FB8 <@t> PALMSG30.us.schp.com>
Content-Type: text/plain
Liz,
Our researchers often freeze tissue for later study. They use the
plastic disposable embedding molds. These can be used with the dry ice as
John Kiernan suggested. There is a thin layer of OCT placed in bottom of
mold then the tissue is oriented in this with additional OCT to fill the
mold. The mold can be labeled with a permanent felt tip pen. Each specimen
is then placed into the labeled sterile sampling bags, packed in dry ice and
shipped to respective location. We have experienced very little distortion
of the tissue due to ice crystals, etc. The testing has worked out okay.
The main tissues were liver, kidney, lung.
You may wish to experiment with some tissues before actually
tackling the research samples to get the right combination for you.
Sharon Osborn
DNAX
ScheringPlough Biopharma
Palo Alto, CA
Hello Histonetters
>
> I have a unique question. We are currently starting to set up
> procedure for collecting samples from a clinical trial. The clinical
> trial involves taking multiple synovial biopsies at a surgery center.
> Since portions of the samples need to be processed for frozen sections
> we wanted to be able to freeze the specimens at the surgery center via
> isopentane cooled liquid nitrogen. We really do not want to have to
> transport the multiple specimens back to the main lab prior to
> freezing due to the time involved it would probably be 1-2 hours post
> biopsy before we could freeze the samples. The surgery center is
> questioning the flammability of the isopentane. Has anyone
> encountered anything like this? Any suggestions would be helpful.
> Thanks in advance.
>
> Liz
>
> Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
> Manager
> Premier Laboratory, LLC
> P.O. Box 18592
> Boulder, Colorado 80308
> Office: (303) 735-5001
> Fax: (303) 735-3540
> liz <@t> premierlab.com
> www.premierlab.com <www.premierlab.com>
>
> Ship to Address:
> Premier Laboratory
> University of Colorado
> MCDB, Room A3B40
> Boulder, Colorado 80309
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------------------------------
Message: 3
Date: Tue, 31 May 2005 15:42:59 -0400
From: "Mitchell, Guy" <Guy.Mitchell <@t> carolinashealthcare.org>
Subject: [Histonet] Enzyme Staining and Billing for Controls
To: "HistoNet Server" <histonet <@t> pathology.swmed.edu>
Message-ID:
<4DCF5CA56B4AA04ABC383DE358BAD3820289100D <@t> dcr-xchg-04.carolinas.org>
Content-Type: text/plain; charset="US-ASCII"
A question has come up concerning charges for muscle enzyme stains. We
stain for Adenylate Deaminase and Phosphofructokinase as well as
negatives for each. I assume they should be treated as controls without
a charge but others disagree.
Guy W. Mitchell
(704) 379-5984
(704) 379-6144 Fax
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Message: 4
Date: Tue, 31 May 2005 15:42:58 -0500
From: "Charles.Embrey" <Charles.Embrey <@t> carle.com>
Subject: RE: [Histonet] Number of Blocks Submitted by PA
To: "Smith Wanda" <Wanda.Smith <@t> HCAhealthcare.com>
Cc: histonet <@t> lists.utsouthwestern.edu
Message-ID:
<C320919A1C432845938BCD8CC3294A30205E69 <@t> EXCHANGEBE1.carle.com>
Content-Type: text/plain; charset="us-ascii"
I have been gone for a week and just got a chance to check my e-mails
and found this question interesting. First, have you asked the PA? I
know it seems like a simple question but you would be surprised at all
the misunderstanding that can be cleared up with a simple question
asked? Please know that I have NEVER met a pathologist that will gladly
read more slides than he thinks are necessary for a good diagnosis on a
routine basis. I gross for seven pathologists and it is tricky to
satisfy all at the same time. Luckily, my pathologists and I here have
great communication and we manage to keep the block count, as well a
gross recut number down. As far as "rules of thumb" we do have certain
guidelines that are basically thought of as "standards of care".
Without knowing other details the best advice I could give is to ask the
PA.
Charles Embrey PA(ASCP)
Urbana IL
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Smith
Wanda
Sent: Tuesday, May 24, 2005 7:56 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Number of Blocks Submitted by PA
Good Morning Histonetters,
I have a question for you this morning...Are there written standards or
"Rules of Thumb" regarding number of blocks that should be submitted on
certain tissues? The reason I ask is, "it seems to me" that sometimes
the PA submits an extreme number of blocks on some specimens that could
be accomplished with less. I'm not advocating cutting corners or not
performing good medical practice, I just need to know. An example would
be a uterus w/ fibroids, tubes and ovaries which we have gotten up to 24
blocks on. I'm not a PA, but it's interesting to me that when the
Pathologist cut, we get alot less blocks.
Please advise.
Thanks,
Wanda
> Wanda G. Smith, HTL/HT(ASCP)
> Pathology Supervisor
> Trident Medical Center Laboratory Services
> *9330 Medical Plaza Drive, Charleston, SC 29406
> *843-797-4586 *fax 843-797-4296
> *wanda.smith <@t> hcahealthcare.com
>
>
>
_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
------------------------------
Message: 5
Date: Wed, 01 Jun 2005 14:13:15 +1000
From: "Anthony Reilly" <Tony_Reilly <@t> health.qld.gov.au>
Subject: [Histonet] Aquaporin immunohistochemistry
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <s29dc288.035 <@t> health-es2.health.qld.gov.au>
Content-Type: text/plain; charset="us-ascii"
Hello All
I have been requested to be involved in a research study
demonstrating Aquaporin 1 (AQ1, AQP1) using IHC. To
date I have found 2 antibodies available from Chemicon.
My questions are:
1. Are there other commercial suppliers of this Ab.
2. If not, which of the 2 from Chemicon is the preferred
option.
3. Do you realyy need to fix with paraformaldehyde as
stated in most methods, and why?
i would appreciate any advice forwarded.
regards
Tony Reilly
Chief Scientist
Anatomical Pathology
Northside Pathology
Prince Charles Hospital
Rode rd Chermside Q 4032
Australia
Ph: 07 3350 8543
Fax: 07 33508546
tony_reilly <@t> health.qld.gov.au
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------------------------------
Message: 6
Date: Wed, 01 Jun 2005 09:48:19 +0200
From: "christian <@t> eisbo.dk" <christian <@t> eisbo.dk>
Subject: [Histonet] Ki67.
To: histonet <@t> lists.utsouthwestern.edu
Message-ID: <758a26d4ec47df2ab23acf9597f0b69d <@t> eisbo.dk>
Content-Type: text/plain; charset=windows-1252
------------------------------
Message: 7
Date: Wed, 01 Jun 2005 09:50:45 +0200
From: "christian <@t> eisbo.dk" <christian <@t> eisbo.dk>
Subject: [Histonet] Ki67.
To: histonet <@t> lists.utsouthwestern.edu
Message-ID: <634ceeb7bf3c9de7df2bf51acb622dfe <@t> eisbo.dk>
Content-Type: text/plain; charset=windows-1252
Hey users of histonet.
I am trying to stain kidneys for ki67!
The sections have been in alcohol for 1 month and are now in parafin.
The antibody I am using should be good enough.
Does anyone have experience with this???
best regards. Christian EIsbo.
------------------------------
Message: 8
Date: Wed, 1 Jun 2005 10:29:19 +0200
From: "Bruijntjes, J.P." <bruyntjes <@t> voeding.tno.nl>
Subject: RE: [Histonet] Ki67.
To: <christian <@t> eisbo.dk>
Cc: Histonet <@t> lists.utsouthwestern.edu
Message-ID:
<3B070848E7C2204F9DEB8BCFD767728001079DC6 <@t> ntexch1.voeding.tno.nl>
Content-Type: text/plain; charset="us-ascii"
Hi Christian
Yes I do. May be it depends on the antibody you use. We use a rabbit
monoclonal ki67 from Neomarkers (Labvision). Tissues fixed in alcohol
don't react with the antibody using HIER in citrate buffer (pH 6,0). At
least it didn't in my lab. It does when you use HIER in EDTA (pH 8.0)
and in TRIS (pH 10).
Hopes that will help you
Joost Bruijntjes
TNO Quality of Life
Zeist
Holland
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of
christian <@t> eisbo.dk
Sent: woensdag 1 juni 2005 9:51
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Ki67.
Hey users of histonet.
I am trying to stain kidneys for ki67!
The sections have been in alcohol for 1 month and are now in parafin.
The antibody I am using should be good enough.
Does anyone have experience with this???
best regards. Christian EIsbo.
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http://lists.utsouthwestern.edu/mailman/listinfo/histonet
This e-mail and its contents are subject to the DISCLAIMER at http://www.tno.nl/disclaimer/email.html
------------------------------
Message: 9
Date: Wed, 1 Jun 2005 10:43:57 +0200
From: "Vincent Martin" <vincent.martin5 <@t> laposte.net>
Subject: [Histonet] visualization of muscle damage with light
microscopy ?
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <002701c56686$0d65e6c0$0a4a7c8b <@t> physiologie8>
Content-Type: text/plain; charset="iso-8859-1"
Dear list members,
I would like to observe the effect of lengthening contractions on
ultrastructural properties of rat tibialis anterior muscle. Is is possible
to observe ultrastructural abnormalities with light microscopy with a x100
maginification ? If not, which are the minimal requirements to observe such
abnormalities ?
Thanks for your replies
Best regards,
Vincent Martin
UPRES-EA 3285 Déterminants Physiologiques de l'Activité Physique
Faculté des Sciences du Sport de Marseille
Université de la Méditerranée
BP910 - 163, avenue de Luminy
13288 Marseille cedex 09
France
Phone : +33 (0)4-91-82-83-78
Fax : +33 (0)4-91-82-83-75
vincent.martin <@t> staps.univ-mrs.fr
<outbind://13/www.physiologie.staps.univ-mrs.fr>
www.physiologie.staps.univ-mrs.fr
------------------------------
Message: 10
Date: Wed, 01 Jun 2005 07:05:40 -0400
From: shawnster73 <@t> aol.com
Subject: [Histonet] Histology of Frozen Tissue
To: histonet <@t> lists.utsouthwestern.edu
Message-ID: <8C734B0FB191BFF-620-23897 <@t> FWM-D40.sysops.aol.com>
Content-Type: text/plain; charset="us-ascii"
What would be the histological effect of allowing frozen OCT embedded tissue to come up to a temperature of 0 or -5oC??
------------------------------
Message: 11
Date: Wed, 1 Jun 2005 08:08:46 -0400
From: "Bobbie Boyce" <bboyce <@t> NEMOURS.ORG>
Subject: [Histonet] BK Virus
To: "HistoNet Server" <histonet <@t> pathology.swmed.edu>
Message-ID:
<6E41111281623B4B8A9AB8F9A7EA343737BFB5 <@t> wlmmsx02.nemours.org>
Content-Type: text/plain; charset=iso-8859-1
I'm having problems locating a FFPE control for BKV. Can anyone tell me
where I can get them?
Bobbie Boyce
duPont Hospital for Children
Wilmington, DE
------------------------------
Message: 12
Date: Wed, 01 Jun 2005 08:10:12 -0400
From: "WAYNE HOLLAND" <jackdodo <@t> msn.com>
Subject: [Histonet] HPV I and II Control Blocks
To: Histonet <@t> lists.utsouthwestern.edu
Message-ID: <BAY107-F21A1094BDF44C14E375105C6050 <@t> phx.gbl>
Content-Type: text/plain; format=flowed
Hello All,
Help, can anyone give assistance to getting some blocks for HPV I and HPV II
for controls. We are going to start In-Situ, we are in the testing phase.
One block from and HPV I (LOW) and one block from HPV II (HIGH) would be
extremely helpful. We would be willing to trade, Thanks in advance.
------------------------------
Message: 13
Date: Wed, 01 Jun 2005 08:44:09 -0500
From: "Corazon D. Bucana" <bucana <@t> audumla.mdacc.tmc.edu>
Subject: Re: [Histonet] mouse endothelial cells in brain
To: "Donin, Nick (NIH/NCI)" <doninn <@t> mail.nih.gov>
Cc: "'histonet <@t> lists.utsouthwestern.edu'"
<histonet <@t> lists.utsouthwestern.edu>
Message-ID: <5.1.1.6.0.20050601084009.042c9c48 <@t> audumla.mdacc.tmc.edu>
Content-Type: text/plain; charset="us-ascii"; format=flowed
The only time we got good staining is on frozen sections using anti-mouse
CD31 from either Pharmingen or Serotec in both immunofluorescence and DAB
or AEC staining.
At 11:54 AM 5/31/2005 -0400, you wrote:
>Histonetters,
>
>This is my first posting, so I'm excited that someone out there might be
>able to help me.
>
>
>I am trying to stain endothelial cells in brain tumors in formalin fixed
>paraffin embedded mouse tissues. I have tried several antibodies, and none
>of them seem to work. I am consistently getting high background and no true
>staining. I have used the following antibodies:
>
>
>
>BD Pharmigen anti-mouse CD31 (PECAM-1) cat #557355
>
>Santa Cruz CD31 (PECAM-1) cat # sc-1506 (I have seen postings that this
>works, but my protocol didn't produce good results)
>
>Cedarlane mouse CD31 cat#CL8930 AP
>
>Cymbus Biotechnology anti-ms CD31 cat# CBL1337
>
>
>
>The protocol I use is shown below.
>
>
>
>
>
>I'm confident that out of these antibodies, at least one will be able to
>stain the endothelial cells in these tumors, but I think that my protocol
>may be the problem. Could someone possibly suggest and antibody or protocol
>that would work for me? Thanks so much for your help, much appreciated.
>
>
>
>Nick Donin
>
>CRTA
>
>Neuro-Oncology Branch
>
>National Cancer Institute
>
>National Institutes of Health
>
>9000 Rockville Pike
>
>Building 35, Room 2B-203
>
>Bethesda, MD 20892
>
>
>
>_______________________________________________
>Histonet mailing list
>Histonet <@t> lists.utsouthwestern.edu
>http://lists.utsouthwestern.edu/mailman/listinfo/histonet
------------------------------
Message: 14
Date: Wed, 1 Jun 2005 10:08:33 -0400
From: "Donin, Nick (NIH/NCI)" <doninn <@t> mail.nih.gov>
Subject: [Histonet] staining with Solvent Blue 38
To: "'histonet <@t> lists.utsouthwestern.edu'"
<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<16A0583FB1644E4DB8C0A0265028B6FD02B4E739 <@t> nihexchange13.nih.gov>
Content-Type: text/plain
Histonetters,
I'm trying to stain the corpus callosum of formalin fixed paraffin embedded
mouse brains using Solvent Blue 38. Could someone suggest a successful
protocol? Thanks.
Nick Donin
CRTA
Neuro-Oncology Branch
National Cancer Institute
National Institutes of Health
9000 Rockville Pike
Building 35, Room 2B-203
Bethesda, MD 20892
------------------------------
Message: 15
Date: Wed, 01 Jun 2005 11:23:20 -0400
From: Luis Chiriboga <Luis.Chiriboga <@t> med.nyu.edu>
Subject: RE: [Histonet] mouse endothelial cells in brain
To: "Corazon D. Bucana" <bucana <@t> audumla.mdacc.tmc.edu>, "Donin, Nick
(NIH/NCI)" <doninn <@t> mail.nih.gov>
Cc: histonet <@t> lists.utsouthwestern.edu
Message-ID: <KFEIIJOCLBABEKFDAEHPGEIKFAAA.Luis.Chiriboga <@t> med.nyu.edu>
Content-Type: text/plain; charset=us-ascii
I have also used the pharmingen cd31 by frozen in mouse brain, muscle, and
skin. It works extremely well in fresh system. FFPE on the other hand is
much trickier. It works but very inconsistently, fixation and tissue
processing are the key variables. Once you have obtained staining in FFPE,
you need to follow the same sacrificing (perfusion etc..) grossing,
processing procedure with "religious fanaticism" (no offense to
anyone.....)
L
____________________________________
Luis Chiriboga Ph.D.
NYU Cancer Institute and
Bellevue Hospital Center
New York University School Of Medicine
Department Of Pathology 4W27
462 First Avenue
New York, N.Y. 10016
W(212) 562-4667.
F(212) 263-2041
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu]On Behalf Of Corazon
D. Bucana
Sent: Wednesday, June 01, 2005 9:44 AM
To: Donin, Nick (NIH/NCI)
Cc: 'histonet <@t> lists.utsouthwestern.edu'
Subject: Re: [Histonet] mouse endothelial cells in brain
The only time we got good staining is on frozen sections using anti-mouse
CD31 from either Pharmingen or Serotec in both immunofluorescence and DAB
or AEC staining.
At 11:54 AM 5/31/2005 -0400, you wrote:
>Histonetters,
>
>This is my first posting, so I'm excited that someone out there might be
>able to help me.
>
>
>I am trying to stain endothelial cells in brain tumors in formalin fixed
>paraffin embedded mouse tissues. I have tried several antibodies, and none
>of them seem to work. I am consistently getting high background and no
true
>staining. I have used the following antibodies:
>
>
>
>BD Pharmigen anti-mouse CD31 (PECAM-1) cat #557355
>
>Santa Cruz CD31 (PECAM-1) cat # sc-1506 (I have seen postings that this
>works, but my protocol didn't produce good results)
>
>Cedarlane mouse CD31 cat#CL8930 AP
>
>Cymbus Biotechnology anti-ms CD31 cat# CBL1337
>
>
>
>The protocol I use is shown below.
>
>
>
>
>
>I'm confident that out of these antibodies, at least one will be able to
>stain the endothelial cells in these tumors, but I think that my protocol
>may be the problem. Could someone possibly suggest and antibody or
protocol
>that would work for me? Thanks so much for your help, much appreciated.
>
>
>
>Nick Donin
>
>CRTA
>
>Neuro-Oncology Branch
>
>National Cancer Institute
>
>National Institutes of Health
>
>9000 Rockville Pike
>
>Building 35, Room 2B-203
>
>Bethesda, MD 20892
>
>
>
>_______________________________________________
>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: 16
Date: Wed, 01 Jun 2005 15:24:32 +0000
From: "CHRISTIE GOWAN" <christiegowan <@t> msn.com>
Subject: [Histonet] Storage of Frozen Section slides
To: histonet <@t> lists.utsouthwestern.edu
Message-ID: <BAY107-F337D0412CFEA59F127DF8DAE050 <@t> phx.gbl>
Content-Type: text/plain; format=flowed
I know this subject has been tossed around before, but I wonder if Patsy
Ruegg or Chris Van der Loos or anyone else for that matter would share with
me their protocol for storing frozen slides. The company I work for sells
frozen TMA slides. We cut sections as requested by clients and mail them out
on dry ice. We have found that after going into the block 10-12 times we
start to see artifact. Pre-cutting slides would be much better for the life
of the block. Has anyone tried storing the slides in a vacuum sealed (food
saver) bag? Any help would be appreciated.
Thanks,
Christie Gowan HT (ASCP)
------------------------------
Message: 17
Date: Wed, 01 Jun 2005 11:18:17 -0400
From: "Vinnie Della Speranza" <dellav <@t> musc.edu>
Subject: Re: [Histonet] Will brain tissue shrink even more if
storedin70%alcoholprior to processing?
To: <jkiernan <@t> uwo.ca>
Cc: jchladny <@t> cvm.uiuc.edu, histonet <@t> lists.utsouthwestern.edu,
clarissabush <@t> sbcglobal.net
Message-ID: <s29d997c.080 <@t> cl.musc.edu>
Content-Type: text/plain; charset=US-ASCII
I hadn't considered the possibility of buffer salt crystals forming the vacuoles observed by the author and I don't know if she is on this list and would care to comment but this is an interesting premise worth investigating. I would think that a thorough water wash prior to going into alcohol would help to clear this up.
thanks for the feedback.
Vinnie
Vinnie Della Speranza
Manager for Anatomic Pathology Services
Medical University of South Carolina
165 Ashley Avenue Suite 309
Charleston, SC 29425
Ph: 843-792-6353
fax: 843-792-8974
>>> John Kiernan <jkiernan <@t> uwo.ca> 05/27/05 04:27PM >>>
In the HistoLogic May 2000 paper the duration of the
initial formaldehyde fixation is not stated; only that
the brains were "previously well fixed in neutral
buffered formalin." Frequently specimens are not
fixed for long enough to make them resistant to
bad effects of solvents etc.
It's also seems from the paper that the test pieces of
brain were passed directly from the buffered fixative
into 70% alcohol. This causes precipitation of
sodium phosphate in the tissue (see Freida Carson
1996 "Histotechnology" 2nd edn, p.27). The control
specimens in the HistoLogic paper were passed from
buffered formalin to 60% ethanol (which safely
extracts the phosphate buffer salts). Could the
holes in the white matter be made by buffer salt
crystals rather than forming slowly during storage
in 70% ethanol?
Just a thought!
--
-------------------------------
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London, Canada N6A 5C1
kiernan[AT]uwo.ca
http://publish.uwo.ca/~jkiernan/
http://instruct.uwo.ca/anatomy/530/index.htm
_______________________________
Vinnie Della Speranza wrote:
>
> You may wish to look at an article authored by Jane Chladny, published in HistoLogic, May 2000, entitled "Artifact in Tissues Held in 70% Ethanol"
>
> the author reported the appearance of 'vacuole' artifact in nervous tissues observed in a variety of mammalian tissues after storage in 70% ethanol.
>
> you can access the article by selecting the link for HistoLogic at www.sakuraus.com
>
> CM Bush wrote:
> >
> > Dear Histonet,
> >
> > Hello, here is my first post to the list, thank you in advance for your help.
> >
> > Summary:
> > Having problems with mouse and human brain tissue shrinkage durning processing, but would like to better preserve antigens, concerned storage of tissue 70% ethanol will shrink tissue even further:
> >
> > We have lots of brain specimens, stored in formalin for a long time (months up to 10 years), both human and mouse. We perform immunohistochemistry on paraffin embedded brain. Of course, there is the massive over fixation. Also the tissue shrinks a lot during processing.
> >
> > In my last lab, after brain tissue had been fixed in formalin for 7 days we would store the tissue in 70% ethanol. I'd like to start storing the brain tissue I work with now in 70%, but I'm worried about the tissue possibly shinking too much, as the tissue already seems to shrink by greater than 60% after processing.
> >
> > (Previously, we would gradually increase the alcohols, 30% for 1 hour, then 50% for an hour in a bucket, room temperature on a rocker table, then put the cassettes into a bucket of 70% and store at room temp or 4C- does this process help any with controling the shrinkage factor?)
> >
> > Maybe this is a little bit long...thank you very much for your time.
> >
> > CM Bush
> ---------
>
> _______________________________________________
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>
> Vinnie Della Speranza
> Manager for Anatomic Pathology Services
> Medical University of South Carolina
> 165 Ashley Avenue Suite 309
> Charleston, SC 29425
> Ph: 843-792-6353
> fax: 843-792-8974
>
> _______________________________________________
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> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
------------------------------
Message: 18
Date: Wed, 1 Jun 2005 11:35:22 -0400
From: "Connolly, Brett M" <brett_connolly <@t> merck.com>
Subject: RE: [Histonet] mouse endothelial cells in brain
To: "'Corazon D. Bucana'" <bucana <@t> audumla.mdacc.tmc.edu>, "Donin, Nick
(NIH/NCI)" <doninn <@t> mail.nih.gov>
Cc: "'histonet <@t> lists.utsouthwestern.edu'"
<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<D9A95B4B7B20354992E165EEADA3199906BD4F69 <@t> uswpmx00.merck.com>
Content-Type: text/plain
Nick,
Fix the brains in Pharmingen Zinc Tris, process to paraffin and use the
Pharmingen antibody. Works like a charm.
Brett
Brett M. Connolly, Ph.D.
Merck & Co., Inc.
MRL, Imaging Research
WP-44K
PO Box 4
West Point, PA 19486
PH 215-652-2501
fax. 215-993-6803
e-mail. brett_connolly <@t> merck.com
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Corazon D.
Bucana
Sent: Wednesday, June 01, 2005 9:44 AM
To: Donin, Nick (NIH/NCI)
Cc: 'histonet <@t> lists.utsouthwestern.edu'
Subject: Re: [Histonet] mouse endothelial cells in brain
The only time we got good staining is on frozen sections using anti-mouse
CD31 from either Pharmingen or Serotec in both immunofluorescence and DAB
or AEC staining.
At 11:54 AM 5/31/2005 -0400, you wrote:
>Histonetters,
>
>This is my first posting, so I'm excited that someone out there might be
>able to help me.
>
>
>I am trying to stain endothelial cells in brain tumors in formalin fixed
>paraffin embedded mouse tissues. I have tried several antibodies, and none
>of them seem to work. I am consistently getting high background and no
true
>staining. I have used the following antibodies:
>
>
>
>BD Pharmigen anti-mouse CD31 (PECAM-1) cat #557355
>
>Santa Cruz CD31 (PECAM-1) cat # sc-1506 (I have seen postings that this
>works, but my protocol didn't produce good results)
>
>Cedarlane mouse CD31 cat#CL8930 AP
>
>Cymbus Biotechnology anti-ms CD31 cat# CBL1337
>
>
>
>The protocol I use is shown below.
>
>
>
>
>
>I'm confident that out of these antibodies, at least one will be able to
>stain the endothelial cells in these tumors, but I think that my protocol
>may be the problem. Could someone possibly suggest and antibody or
protocol
>that would work for me? Thanks so much for your help, much appreciated.
>
>
>
>Nick Donin
>
>CRTA
>
>Neuro-Oncology Branch
>
>National Cancer Institute
>
>National Institutes of Health
>
>9000 Rockville Pike
>
>Building 35, Room 2B-203
>
>Bethesda, MD 20892
>
>
>
>_______________________________________________
>Histonet mailing list
>Histonet <@t> lists.utsouthwestern.edu
>http://lists.utsouthwestern.edu/mailman/listinfo/histonet
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Message: 19
Date: Wed, 01 Jun 2005 12:19:17 -0400
From: Tim Wheelock <twheelock <@t> mclean.harvard.edu>
Subject: [Histonet] SOLVENT BLUE 38 ON PARAFFIN EMBEDDED MOUSE BRAIN
To: "'histonet <@t> lists.utsouthwestern.edu'"
<histonet <@t> lists.utsouthwestern.edu>
Message-ID: <429DE005.1050905 <@t> mclean.harvard.edu>
Content-Type: text/plain; charset=windows-1252; format=flowed
Hi Nick:
I use Solvent Blue 38 (also known as Luxol Fast Blue) on formalin-fixed,
paraffin embedded human brain, but have also seen it work well on mouse
brain.
The protocol is as follows. This particular recipe combines the myelin
stain with an H+E, but it can be combined with a Nissl stain instead or
simply by itself.
Hope this helps.
Tim Wheelock
Harvard Brain Tissue Resource Center
McLean Hospital
617-855-3592
* LUXOL FAST BLUE**-HEMATOXYLIN-EOSIN (LHE) STAIN*
* *
1. De-paraffinize 5 micron formalin-fixed sections and take them down to
95% ethanol.
2. Stain in Luxol Fast Blue solution for 2 hours at 60C.
3. Remove and allow to cool for 15 minutes.
4. Remove excess stain in 95% ethanol for 1 minute.
5. Wash in several changes of tap water.
6. Differentiate sections with 2 dips in the reducer solution, then
*immediately* through 4
changes of tap water (10 dips each), then one more change of tap water.
7. Immerse sections in Gill 3 Hematoxylin for 10 minutes.
8. Rinse in several changes of tap water.
9. Differentiate sections in acid alcohol (7 dips).
10. Rinse in 4 changes of tap water (10 dips each) then one more change
of tap water.
11. Blue sections in Scott's Tap Water Substitute for 30 seconds.
12. Wash in 4 changes of tap water for 2 minutes each.
13. Immerse sections in 95% ethanol for 1 minute.
14. Immerse slides in alcoholic Eosin Y for 3 minute.
15. Differentiate in 95% ethanol (4 dips).
16 Dehydrate in first absolute ethanol for 20 dips.
17. Dehydrate in second absolute ethanol for 1 minute, and then clear,
and mount.
*SOLUTIONS*: Luxol Fast Blue: Luxol Fast
Blue...............................................0.1 gram.
10% glacial acetic acid .......................................1 mls.
95% ethanol........................................................100mls.
Reducer:
Hydroquinone....................................................1 gram.
Sodium Sulfite...................................................5 gram.
Distilled water..................................................100 mls.
Hematoxylin Differentiator: 1% Hydrochloric acid in 70% ethanol
*RESULTS*:
1. Myelin: The myelin should be stained blue or greenish blue
2. Nuclei: The nuclear membrane should be sharply delineated in
blue-black with the
nucleoplasm clear except for the chromatin, also being blue-black.
3. Cytoplasm and background should be varying shades of red.
NOTE
1. The thickness of the section, the type of hematoxylin, and all the
times in hematoxylin, eosin, and dehydrating depend upon your requirements.
2. If the section thickness is more than 10 microns, you may want to use
3 or 4 dips in the Reducer, so as to avoid background myelin staining .
3. The so called "Reducer" (because it is used as a reducer in the
Bodian silver protein stain) actually functions as a differentiator for
the myelin stain.
------------------------------
Message: 20
Date: Wed, 1 Jun 2005 12:18:44 -0400
From: Diana McCaig <dmccaig <@t> ckha.on.ca>
Subject: [Histonet] filtering hematoxylin
To: "Histonet (E-mail)" <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <3E5A3F039F0BD8118B4700C00D0020240435BF <@t> CKHA9>
Content-Type: text/plain
How often do you filter commercially prepared Harris Hematoxylin (used on
autostainer)?
------------------------------
Message: 21
Date: Wed, 1 Jun 2005 12:33:58 -0400
From: "Bartlett, Jeanine" <jqb7 <@t> cdc.gov>
Subject: RE: [Histonet] filtering hematoxylin
To: "Diana McCaig" <dmccaig <@t> ckha.on.ca>, "Histonet \(E-mail\)"
<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<CB857F6460D42E4AAEA195054A25406C076162CF <@t> m-ncid-2.ncid.cdc.gov>
Content-Type: text/plain; charset="us-ascii"
We don't filter the hematoxylin we get from Richard-Allan. We replace
it twice a month, but we have a light volume of H&E's.
Jeanine Bartlett, HT(ASCP)
Centers for Disease Control and Prevention
1600 Clifton Road, MS/G-32
Atlanta, GA 30333
(404) 639-3590
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Diana
McCaig
Sent: Wednesday, June 01, 2005 12:19 PM
To: Histonet (E-mail)
Subject: [Histonet] filtering hematoxylin
How often do you filter commercially prepared Harris Hematoxylin (used
on autostainer)?
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End of Histonet Digest, Vol 19, Issue 1
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