[Histonet] RE: Histonet Digest, Vol 24, Issue 25
Featherstone, Annette
AFeatherstone <@t> KaleidaHealth.Org
Fri Nov 18 05:22:11 CST 2005
We use recycled xylene in processing and staining. We have sent samples of
our recycled xylene to be tested for purity and it is actually better than
new.
Annette Featherstone
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu]On Behalf Of
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Sent: Thursday, November 17, 2005 13:02
To: histonet <@t> lists.utsouthwestern.edu
Subject: Histonet Digest, Vol 24, Issue 25
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Today's Topics:
1. QIHC (Elizabeth Chlipala)
2. RE: recycled xylenes (Bauer, Karen)
3. RE: QIHC (James Watson)
4. RE: RE: QIHC change (Smith, Jeffery D. (HSC))
5. cryoprotection (Steven Coakley)
6. am I snap-freezing correctly? (Jacobs,Jennifer (stu))
7. RE: am I snap-freezing correctly? (Favara, Cynthia (NIH/NIAID))
8. Re: Luxol fast blue/cresyl violet stains (Rene J Buesa)
9. Re: recycled xylenes (Rene J Buesa)
10. Re: am I snap-freezing correctly? (John A. Kiernan)
11. RE: QIHC change (Johnson, Teri)
12. Help- triphenyltetrazolium chloride instructions for staining
needed (HCS)
13. Re: Histonet Digest, Vol 24, Issue 23 (JUDIEWHIT <@t> aol.com)
14. biohazard waste removal companies (Ron Martin)
15. RE: aqueous coverslipping medium (C.M. van der Loos)
16. hard tissue distortion (louise renton)
17. CD31 staining histiocytes??? (Histology SLU)
18. CD31 Santa Cruz update (Angela McNabola)
19. RE: E: beta-2- microglobulin (Edwards, R.E.)
20. fixed jaw clamps/ large size specimens (Kinsley, David)
21. Dissecting Microscopes (Bauer, Karen)
22. Re: fixed jaw clamps/ large size specimens (Rene J Buesa)
----------------------------------------------------------------------
Message: 1
Date: Wed, 16 Nov 2005 13:08:53 -0700
From: "Elizabeth Chlipala" <liz <@t> premierlab.com>
Subject: [Histonet] QIHC
To: "'Histonet'" <histonet <@t> pathology.swmed.edu>
Message-ID: <000001c5eae9$91e76df0$a7d48a80 <@t> AMY>
Content-Type: text/plain; charset="us-ascii"
I'm going to have to chime in here. One thing I need to say upfront is
that I served on the BOR when they were developing the QIHC. The BOR
for the histotech exam committee consists of histology technicians,
histotechnologists, MD pathologists and veterinary pathologists, plus
the ASCP staff, so it encompassed a wide variety of individuals and
skill sets, both in research and clinical. These are the individuals
who created the exam and the requirements for the exam. I went to the
BOR guidelines online for the QIHC and as far as I can see that have not
changed. Immunophenotyping has always been a pre-requisite for sitting
for this qualification. This is what BOR requires:
Immunophenotyping in at least one of the following applications
* immunodeficiencies
* immunoproliferative disorders (neoplastic and non-neoplastic
disorders)
* transplantation biopsies
* other immunophenotyping applications
please specify: ______________________
After looking online for the definition of immunophenotyping it is the
following: " IMMUNOPHENOTYPING refers to the technique of identifying
molecules that are associated with lymphoid cells and help to
characterize them". Such as cell surface markers, which I would
characterize broadly as CD markers. I work in research and sat for the
QIHC last year. In research we perform many Immunohistochemical stains
utilizing CD markers to determine what specific cell types are present
in certain disease states. When I filled out my application last year I
selected the forth choice which is "immunophenotyping applications
other" and listed what types of CD markers I had worked with and the
type of projects and applications associated with them. I was able to
sit for the exam and so was my employee. In my opinion many histotechs
in research have the necessary experience since they are performing
Immunohistochemical staining utilizing a wide variety of CD markers. I
just don't see how in working in research you would not have the
pre-requisite to sit for this exam.
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
Ship to Address:
Premier Laboratory
University of Colorado
MCDB, Room A3B40
Boulder, Colorado 80309
------------------------------
Message: 2
Date: Wed, 16 Nov 2005 14:14:06 -0600
From: "Bauer, Karen" <Bauer.Karen <@t> mayo.edu>
Subject: RE: [Histonet] recycled xylenes
To: "Molinari, Betsy" <BMolinari <@t> heart.thi.tmc.edu>,
<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<FAEEA161EC98C6439D4892E161BCE18A1F2309 <@t> LMMAILVS1.ad.lmmhs.org>
Content-Type: text/plain; charset="iso-8859-1"
Hi Betsy,
We use are recycled xylene for both processing and staining and have had no
problems.
Karen L. Bauer HT(ASCP)
Histology Supervisor
Luther Hospital
Eau Claire, WI
________________________________
From: histonet-bounces <@t> lists.utsouthwestern.edu on behalf of Molinari, Betsy
Sent: Wed 11/16/2005 2:00 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] recycled xylenes
Hi,
I am interested in knowing if you have preferences for your recycled
xylenes. Do you use it just for staining, or processing alone. Or use
it for both?
Thank you.
Betsy Molinari HT (ASCP)
Texas Heart Institute
Cardiovascular Pathology
6770 Bertner Ave.
Houston,TX 77030
832-355-6524
832-355-6812 (fax)
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Message: 3
Date: Wed, 16 Nov 2005 12:20:09 -0800
From: "James Watson" <jwatson <@t> gnf.org>
Subject: RE: [Histonet] QIHC
To: "Elizabeth Chlipala" <liz <@t> premierlab.com>,
<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<D8EB530DD5F90B498AAB7BEB68FBED770BCDD5 <@t> EXCHCLUSTER01.lj.gnf.org>
Content-Type: text/plain; charset="us-ascii"
Elizabeth,
I have been trying to find out what the other category entails without
success, this is why I became involved with these discussions to see if
someone on the histo net knew. But I have finally gotten someone at
ASCP to talk to as listed on my last posting. She was not very clear as
to what qualifies and if work on animal tissue is acceptable. So she
wants me to send her a list of what I do that may qualify as
immunophenotyping to determine if I qualify.
I really appreciate your response because it gives me some guidelines as
what I can list as other immunophenotyping applications. Thank you very
much.
James Watson HT, ASCP
Facilities Manager of Histology
GNF, Genomics Institute of the Novartis Research Foundation
Room C015
858-332-4647
jwatson <@t> gnf.org
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of
Elizabeth Chlipala
Sent: Wednesday, November 16, 2005 12:09 PM
To: 'Histonet'
Subject: [Histonet] QIHC
I'm going to have to chime in here. One thing I need to say upfront is
that I served on the BOR when they were developing the QIHC. The BOR
for the histotech exam committee consists of histology technicians,
histotechnologists, MD pathologists and veterinary pathologists, plus
the ASCP staff, so it encompassed a wide variety of individuals and
skill sets, both in research and clinical. These are the individuals
who created the exam and the requirements for the exam. I went to the
BOR guidelines online for the QIHC and as far as I can see that have not
changed. Immunophenotyping has always been a pre-requisite for sitting
for this qualification. This is what BOR requires:
Immunophenotyping in at least one of the following applications
* immunodeficiencies
* immunoproliferative disorders (neoplastic and non-neoplastic
disorders)
* transplantation biopsies
* other immunophenotyping applications
please specify: ______________________
After looking online for the definition of immunophenotyping it is the
following: " IMMUNOPHENOTYPING refers to the technique of identifying
molecules that are associated with lymphoid cells and help to
characterize them". Such as cell surface markers, which I would
characterize broadly as CD markers. I work in research and sat for the
QIHC last year. In research we perform many Immunohistochemical stains
utilizing CD markers to determine what specific cell types are present
in certain disease states. When I filled out my application last year I
selected the forth choice which is "immunophenotyping applications
other" and listed what types of CD markers I had worked with and the
type of projects and applications associated with them. I was able to
sit for the exam and so was my employee. In my opinion many histotechs
in research have the necessary experience since they are performing
Immunohistochemical staining utilizing a wide variety of CD markers. I
just don't see how in working in research you would not have the
pre-requisite to sit for this exam.
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
Ship to Address:
Premier Laboratory
University of Colorado
MCDB, Room A3B40
Boulder, Colorado 80309
_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
------------------------------
Message: 4
Date: Wed, 16 Nov 2005 14:18:09 -0600
From: "Smith, Jeffery D. \(HSC\)" <Jeffery-Smith <@t> ouhsc.edu>
Subject: RE: [Histonet] RE: QIHC change
To: "Morken, Tim - Labvision" <tpmorken <@t> labvision.com>,
<Histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<EA80A7E0A540C44A8A339A3561E35C60063E2627 <@t> PERCHERON.hsc.net.ou.edu>
Content-Type: text/plain; charset="iso-8859-1"
Tim,
I would like to know what your certification is and what qualifies you to
decide what the focus of the ASCP certifications entale? Who put you in
charge of deciding what the sole purpose of the ASCP is?
________________________________
From: histonet-bounces <@t> lists.utsouthwestern.edu on behalf of Morken, Tim -
Labvision
Sent: Wed 11/16/2005 1:04 PM
To: 'Pamela Marcum'; Morken, Tim - Labvision;
histonet <@t> lists.utsouthwestern.edu
Subject: RE: [Histonet] RE: QIHC change
Pam,
I'm not discounting the knowledge researchers have. I just don't think the
ASCP certification route fits research. I pointed out that the ASCP, by
definition, is intended soley for clinical diagnostic labs. It is not the
ASCP's fault that the research institutions are mis-using the ASCP
certification for their own in-house qualification standards. Certainly it
would help researchers if there was a more general certification, but is it
the ASCP's place to do that? I don't know. The ASCP is an organization of
Clinical Pathology based in human diagnostics. Because of that the HT, HTL
and QIHC are all based on human clinical pathology diagnostic work. I'm not
sure the ASCP would be interested in setting up a whole new testing
procedure for research. What if a person only does work in ferrets, or fish,
or snakes? Who looks at tests based on that work? There are many
similarities in procedures, and I'm confident a generalized test could be
designed. It simply a question of whether the ASCP is the organization to do
that. I guess the research techs have to explore this with ASCP. But I don'
think you should blame ASCP for not accomodating research institutions when
that is not what they are concerned with. Remember, there are no regulatory
requirements at all covering the work of research lab techs but there are
regulations covering who can do certain work in the diagnostic lab. ASCP,
again by definition, is only concerned with the diagnostic side. It has
nothing to do with whether one is "better" than another. There is no
"better" in this work, only differences in the work done. I also suggest
that you lobby your institution and show them how the ASCP certification may
not be fully applicable to what you do.
Tim Morken
-----Original Message-----
From: Pamela Marcum [mailto:pmarcum <@t> vet.upenn.edu]
Sent: Wednesday, November 16, 2005 9:56 AM
To: Morken, Tim - Labvision; histonet <@t> lists.utsouthwestern.edu
Subject: RE: [Histonet] RE: QIHC change
Hi Tim and Jamie,
I know Jamie and have for many years and you are right anyone would be
happy to hire him for his experience in clinical or his current research
position. However, it is also true that now research positions are often
asking for at least an HT or even HTL (ASCP) to fill position with
histology as the main focus. Yet we are given a set of criteria for tissue
that often excludes animal research applicants from completing the
practical easily. I took my HT many years ago and I was told that even in
a research position (and I had a BS at the time) it would improve my salary
and increase me to higher level in the university if I got my HT. I was
told not to use animal tissue (1976) as no one reading the exam could
properly read them. Now we have veterinary person there and tissue
requirements can still eliminate some people or make it almost impossible
to complete the practical with out help in procurement. Why should that
happen to some one attempting to improve their position within the
histology community?
My real problem with what you said about the QIHC is that I would also like
to take it and can not qualify either. Yet those of us in research are
often finding the very antibodies and test methods companies and
diagnostics later fight to get or learn. We are exempt in your mind and
ASCP's even though research is what you depend on often for advances. I
have never and will never understand this logic and exempt status for those
of us who chose not to be clinical. We are still often required to have
or get ASCP status as a way to advance and prove we know our field. ASCP
needs to get up to date on the fields it is registering or make new
categories for those of still contribute to clinical advances every year.
Sorry if sounds like I am picking on you Tim. I just don't see how we are
required to be registered on one hand for acceptance (even NSH likes to see
it) and discounted on the other.
Pam Marcum
UPENN Vet School
New Bolton Center
Kennett Square, PA 19384
610-925-6278
At 12:07 PM 11/16/2005, Morken, Tim - Labvision wrote:
>Jamie, It seems from what you say that you are working in a research
>lab. Is that correct? My understanding about the ASCP certification is
>that it is aimed at providing a modicum of proof that a person is
>qualified to work in a medical diagnostic lab. Research labs are not
>considered diagnostic labs. As you imply, a person in a research lab
>will often work on only a limited sample set. Therefore, it is
>meaningless to apply the the ASCP standard to research people.
>
> If you are planning to move into the diagnostic field, then I'll bet
>you could easily find a job in a diagnostic lab, get the experience,
>and qualify to take the test. It may be that some diagnostic labs have
>a suggested requirement to be ASCP certified as a QIHC, but the vast
>majority would be happy to find someone with the experience you
>outline, even if they had not previously worked in a diagnositc lab.
>
>Tim Morken
>Lab Vision - Neomarkers
>www.labvision.com
>
>Free webhosting for US State Histotechnology Societies:
>http://www.labvisioncorp.com/demowebsite/index.cfm
>
>-----Original Message-----
>From: histonet-bounces <@t> lists.utsouthwestern.edu
>[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of James
>Watson
>Sent: Wednesday, November 16, 2005 7:28 AM
>To: novanity <@t> nc.rr.com; histonet <@t> lists.utsouthwestern.edu
>Subject: RE: [Histonet] RE: QIHC change
>
>
>This is my point. With the requirements listed below someone with 25
>years of experience doing immuno (single, double, triple antibody
>staining, making own antibodies, and in situ Hybridization: all with
>and without using kits, all with and without using an automated
>stainer) is not qualified for this certification if they work in a
>research facility where immunophenotyping is not done. There is no
>system of doing it on your own to prove that you have the capability to
>do immunophenotyping in order to fullfil this requirement. I guess it
>is time to start harrassing ASCP about the unfairness of this system.
>
> >From almost always sunny San Diego
>Jamie
>
> -----Original Message-----
> From: histonet-bounces <@t> lists.utsouthwestern.edu on behalf of
>novanity <@t> nc.rr.com
> Sent: Wed 11/16/2005 5:55 AM
> To: histonet <@t> lists.utsouthwestern.edu
> Cc:
> Subject: [Histonet] RE: QIHC change
>
>
>
> James, to qualify for the qualification you take the 50
>question test
> and submit an employer reference form + of course satisfy one of
the
> three routes. There is no practical to submit anymore. I
>think that is
> what you are asking. It seems to me that it wouldn't matter about
> specificity antigens/markers or what diseases or human cells.
>There is
> no requirement other than what is requested on the employer
>reference
> form which you can't see the details until you order and
>receive your
> packet. Is it possible for anyone to post a copy of the employer
> reference form. From the ASCP website this is what it says "
>
> Qualification in Immunohistochemistry
> Experience requirements
>
> Applicants must have experience in the following areas
>
> * Immunohistochemical and Immunofluorescence Preparation
> All of the following should have been performed by the
>applicant
> o staining technique
> o selection of proper control material
> o titration of immunologic reagents
>
> * Immunophenotyping
> in at least one of the following applications
> o immunodeficiencies
> o immunoproliferative disorders (neoplastic and
>non-neoplastic
> disorders)
> o transplantation biopsies
> o other immunophenotyping applications
> please specify: ______________________
>
> * Quality Assurance
> The applicant should have participated in Quality Assurance
> related to all of the following
> o specimen fixation, processing, microtomy
> o reagent selection, preparation, storage, disposal
> o method selection, validation, documentation
> o quality control
> o safety
>
> " This is the experience which I am assuming is only
>documented for the
> ASCP through the employer reference form, hence if you only do
>A and C
> and not B you can't qualify unless your employer is dishonest on
the
> form. Because even if you crosstrain into what I assume is flow
> cytometry but don't actually work it day to day as part of
>your job you
> do not qualify because you have not had experience doing it for a
> minimun of 12 months. As for research, same thing if you do
>all of it
> every day then your good to go. If not it is a grey or is it
>gray area
> that I'm looking more information/details on. In the past you
> qualified your work with different immuno stains as a practical ,
I
> don't remember there being a flow requirement. Maybe I'm wrong
but
> anyone have this info I'm looking for.
>
> G Hurlburt HT(ASCP)
> sunny and warm NC
>
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>
>
>_______________________________________________
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------------------------------
Message: 5
Date: Wed, 16 Nov 2005 12:26:44 -0800 (PST)
From: Steven Coakley <sjchtascp <@t> yahoo.com>
Subject: [Histonet] cryoprotection
To: Histonet <@t> lists.utsouthwestern.edu
Message-ID: <20051116202644.45169.qmail <@t> web90201.mail.scd.yahoo.com>
Content-Type: text/plain; charset=iso-8859-1
How are techs freezing their fixed, 30% infiltrated tissue. Right now we
freeze the tissue (liver) using OCT placed in a small freezing cup and float
on liquid nitrogen.
They section great but the cellular detail is only fair. Does anyone have
really good result cryoprotecting tissue.
Steve
---------------------------------
Yahoo! FareChase - Search multiple travel sites in one click.
------------------------------
Message: 6
Date: Wed, 16 Nov 2005 15:31:07 -0500
From: "Jacobs,Jennifer \(stu\)" <JJacobs <@t> student.uchc.edu>
Subject: [Histonet] am I snap-freezing correctly?
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <9F866D418736DD408F43059FF7FC697203B34973 <@t> itnsa.uchc.edu>
Content-Type: text/plain; charset="Windows-1252"
Hi,
I am snap freezing brain tissue for sectioning (8uM) on cryostat (-25C).
When I snap freeze, I put the brain hemisphere on a piece of aluminum foil
(boat) and let it float on the LN2. Most times, some LN2 comes in direct
contact with the brain tissue as the boat is not leak-free. Is this bad?
Should I make sure the LN2 never comes in contact with the brain?
The problem I've been noticing is that, right off the cryostat, my tissue
looks "hole-y". This just gets worse as I proceed with the IHC stain (CD-31
Ab to see vessels) and dehydration + xylene steps. By the xylene steps the
holes are so stretched out that I can't even recognize vessels in the
tissue.
I am not sure whether the holes arise from the brain extraction procedure
(ie: snap freezing) or from the sectioning. But the person who sections has
been doing it for years and has great experience with the cryostat.
Any ideas/suggestions would be great! Thanks a lot!
Jenn
UConn Health Center
Dept Pharmacology
Farmington, CT
------------------------------
Message: 7
Date: Wed, 16 Nov 2005 15:47:08 -0500
From: "Favara, Cynthia \(NIH/NIAID\)" <cfavara <@t> niaid.nih.gov>
Subject: RE: [Histonet] am I snap-freezing correctly?
To: "Jacobs,Jennifer \(stu\)" <JJacobs <@t> student.uchc.edu>,
<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<A985B7D45D355D4EB363288E717394375B805C <@t> NIHCESMLBX5.nih.gov>
Content-Type: text/plain; charset="us-ascii"
Look in the archives for the protocol for freezing over isopentane by
Gayle Callis. This is what I find this better than LN for morphology -
ditto for the cryoprotected specimens.
c
Cynthia Favara
NIAID/NIH/RML/LPVD
903 South 4th Street
Hamilton, MT 59840
406-363-9317
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-----Original Message-----
From: Jacobs,Jennifer (stu) [mailto:JJacobs <@t> student.uchc.edu]
Sent: Wednesday, November 16, 2005 1:31 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] am I snap-freezing correctly?
Hi,
I am snap freezing brain tissue for sectioning (8uM) on cryostat (-25C).
When I snap freeze, I put the brain hemisphere on a piece of aluminum
foil (boat) and let it float on the LN2. Most times, some LN2 comes in
direct contact with the brain tissue as the boat is not leak-free. Is
this bad? Should I make sure the LN2 never comes in contact with the
brain?
The problem I've been noticing is that, right off the cryostat, my
tissue looks "hole-y". This just gets worse as I proceed with the IHC
stain (CD-31 Ab to see vessels) and dehydration + xylene steps. By the
xylene steps the holes are so stretched out that I can't even recognize
vessels in the tissue.
I am not sure whether the holes arise from the brain extraction
procedure (ie: snap freezing) or from the sectioning. But the person who
sections has been doing it for years and has great experience with the
cryostat.
Any ideas/suggestions would be great! Thanks a lot!
Jenn
UConn Health Center
Dept Pharmacology
Farmington, CT
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------------------------------
Message: 8
Date: Wed, 16 Nov 2005 12:56:17 -0800 (PST)
From: Rene J Buesa <rjbuesa <@t> yahoo.com>
Subject: Re: [Histonet] Luxol fast blue/cresyl violet stains
To: judi.ford <@t> jax.org, histonet <@t> lists.utsouthwestern.edu
Message-ID: <20051116205617.42144.qmail <@t> web61218.mail.yahoo.com>
Content-Type: text/plain; charset=iso-8859-1
Judi:
When you say "5 slides from the same animal" do you mean slides from
different blocks from the same animal or slides from the same block.
If the slides are from different blocks then the problem could reside in
the blocks and how they were treated. If all the slides are from the same
block then the difference could reside in the configuration the slides were
placed in the staining jar (although this explanation could be far fetched).
I don't think that the differences could be due to the differentiation
step because you say that after differentiation the slides are placed in
distilled water, had they been "waiting" for the result of one slide to stop
the differentiation on the others that could be the cause.
Now, if the 37Celsius incubation is done in an oven and the oven does not
have a good air circulation it could be that one area in the staining jar
could have a difference in temperature at least during some time, although
that could be compensated during the long incubation period. So I really
don't know what to atribute the problem to.
Not exactly these, but the delay itself, prompted us to eliminate this
protocol and go to one using microwaves (MW) as follows:
1- dewax and take sections to absolute ethanol (EthOL)
2-place slides in plastic Coplin jar and add 95% EThOL
3- heat slides in the 95% EthOL for 15 secs. at 0.95 kcal ("Level 5" of
our 480 W MW oven which was equivalent to 266 Watts)
4- dump the heated 95% EthOL (used only to heat the slides) and substitute
it with 40 mL of Luxol Fast Blue (LFB). Heat at the same level (266 Watts)
for 20 secs (=1.27 kcal).
5- transfer the Coplin jar with the slides to a water bath at 60Celsius
during 5 minutes.
6- Wash slides in distilled water.
7- Differentiate the slides , one at a time, with 0.5%aq. lithium
carbonate solution until no more blue color runs off the slides (not
necessary under the microscope)
8- Transfer the slides to distilled water.
9- Dump the dist. water and add the Cresyl Echt Violet (CEV) and heat in
the MW at full power (480 Watts) for 20 secs.(=2.3 kcal).
10-Transfer the slides to the water bath at 60Celsius for 5 minutes.
11- Dehydrate QUICKLY, clear and mount as usual.
With this procedure we did not have any problems and it could be completed
in about 30 minutes. Perhaps you could care to try this protocol to find out
if the problem persists.
This is how we did it in our lab. Hope this will help!
Rene J.
judi.ford <@t> jax.org wrote:
Hi,
I have a question concerning the lfb/cv stain. This is the situation we have
in our lab. The tissue we are working on is Bouin's fixed mouse brain and
spinal cord (cord is left in the vertebrae). The spinal cord may be left in
Bouin's for an extended period for decalcification (two-three weeks). The
trimmed tissue is then sent to our lab where we rinse it for a day before
processing on our VIP processors. The next day we embed the tissue and then
it is cut by one of our technicians and stained. This is our staining
technique: deparaffinize to 95% alcohol and place in alcoholic luxol fast
blue overnight at 37 degrees. The next morning it is rinsed twice in 95%
alcohol and twice in distilled water. The slides are separated into racks
containing either brain or spinal cord for differentiation. Slides are
dipped in 70% alcohol for 20 seconds then lithium carbonate for 20 seconds.
This is followed by two rinses in distilled water. Then checked
microscopically before determining if
differentiation is complete or not. If not, then the slides go another
round through 70% and lithium for a few more seconds. Once this part is done
the slides are put into a warmed cresyl violet solution (we add 10% glacial
acetic acid, 10 drops for every 100mls of stain) for 4-6 minutes then rinsed
in 95% alcohol three times. Finally they are dehydrated, cleared and
coverslipped.
The problem comes when we have 5 slides from the same animal, all processsed
and stained together, where in one slide the cresyl violet works but in the
other 4 it doesnt' work. Every slide is treated exactly the same, yet we
have this difference. Any ideas on what may be happening? We've discussed
decalcification and possible lithium effects on the cresyl violet. We're at
a loss and our pathologist is very determined to discover the solution for
this problem.
Thank you advance for any ideas you may send our way..........We greatly
appreciate your help!
Judi Ford
HIstotechnologist
HIstopathology & Microscopy
The Jackson Laboratory
600 Main St.
Bar Harbor, Me 04609
207-288-6193
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Message: 9
Date: Wed, 16 Nov 2005 13:01:46 -0800 (PST)
From: Rene J Buesa <rjbuesa <@t> yahoo.com>
Subject: Re: [Histonet] recycled xylenes
To: "Molinari, Betsy" <BMolinari <@t> heart.thi.tmc.edu>,
histonet <@t> lists.utsouthwestern.edu
Message-ID: <20051116210146.28437.qmail <@t> web61224.mail.yahoo.com>
Content-Type: text/plain; charset=iso-8859-1
Betsy:
We used to recycle xylene and used for everything. Since the recovery is
not 100% when we had to buy new we preferred to use it in the staining and
coverslippin instruments.
Rene J.
"Molinari, Betsy" <BMolinari <@t> heart.thi.tmc.edu> wrote:
Hi,
I am interested in knowing if you have preferences for your recycled
xylenes. Do you use it just for staining, or processing alone. Or use
it for both?
Thank you.
Betsy Molinari HT (ASCP)
Texas Heart Institute
Cardiovascular Pathology
6770 Bertner Ave.
Houston,TX 77030
832-355-6524
832-355-6812 (fax)
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Message: 10
Date: Wed, 16 Nov 2005 16:07:06 -0500
From: "John A. Kiernan" <jkiernan <@t> uwo.ca>
Subject: Re: [Histonet] am I snap-freezing correctly?
To: "Jacobs,Jennifer (stu)" <JJacobs <@t> student.uchc.edu>
Cc: histonet <@t> lists.utsouthwestern.edu
Message-ID: <437B9F7A.7E99AF0F <@t> uwo.ca>
Content-Type: text/plain; charset=us-ascii
The more holey than righteous appearance that you
describe is typical of slowly frozen tissue - especially
if it hasn't been cryoprotected.
When liquid nitrogen comes into contact with an object
it boils, and the resulting layer of nitrogen gas has
an insulating effect that slows down the cooling process.
For that reason it is preferable to freeze specimens
quickly by immersing in a liquid that has been pre-cooled
to a low temperature. The one most often used is
isopentane. Put some in a small metal can, and stand the
can in liquid nitrogen. Stir the isopentane with a
lollipop stick. When it starts to thicken the temperature
is approaching its freezing point (-160C) and the specimen
(with or without aluminium foil) can be dropped in.
Another method is to bring a fairly massive block of
metal to liquid nitrogen temperature (-196C) and slap the
specimen onto the surface of the block.
You will probably get plenty of replies. There has been
much Histonetting about quick freezing methods over the
years!
--
-------------------------------
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
_______________________________
"Jacobs,Jennifer (stu)" wrote:
>
> Hi,
>
> I am snap freezing brain tissue for sectioning (8uM) on cryostat (-25C).
> When I snap freeze, I put the brain hemisphere on a piece of aluminum foil
(boat) and let it float on the LN2. Most times, some LN2 comes in direct
contact with the brain tissue as the boat is not leak-free. Is this bad?
Should I make sure the LN2 never comes in contact with the brain?
>
> The problem I've been noticing is that, right off the cryostat, my tissue
looks "hole-y". This just gets worse as I proceed with the IHC stain (CD-31
Ab to see vessels) and dehydration + xylene steps. By the xylene steps the
holes are so stretched out that I can't even recognize vessels in the
tissue.
>
> I am not sure whether the holes arise from the brain extraction procedure
(ie: snap freezing) or from the sectioning. But the person who sections has
been doing it for years and has great experience with the cryostat.
>
> Any ideas/suggestions would be great! Thanks a lot!
>
> Jenn
>
> UConn Health Center
> Dept Pharmacology
> Farmington, CT
>
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
------------------------------
Message: 11
Date: Wed, 16 Nov 2005 15:36:26 -0600
From: "Johnson, Teri" <TJJ <@t> Stowers-Institute.org>
Subject: [Histonet] RE: QIHC change
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<C28BAF593DC3314E9C0F3A50191C2E78016CB90D <@t> EXCHKC03.stowers-institute.org>
Content-Type: text/plain; charset="us-ascii"
James, et al,
Thank you for bringing lively discussion in an area I believe needs it.
I have worked for many years in clinical histology labs and now work in
the research environment. I have to admit, once I heard they had moved
this exam to a 50-question test, I signed up. Previously, I knew it
would be nearly impossible for me to do the practical exam with human
tissue samples since we don't have any here. I took the exam last week
and have to say that while most of the questions were based on general
IHC knowledge, a couple of questions regarded clinical samples (cytology
specimens) not usually done in research, or were geared toward estrogen
receptor-type immunostaining. Most of my issues with the exam related
to the wording of the question, or the choices given (I didn't always
agree with them, and one was ambiguous). I'm glad I now have a contact
to whom I can give my opinion of the exam, thanks James!
My conclusion was, the exam can be taken successfully by research techs
with a strong background in IHC, and the clinical-only questions they
will miss will still give them enough margin to pass. (Provided, of
course, they meet the requirements.) I agree as well that the
immunophenotyping requirement be phased out or reworded to show
knowledge of typical staining patterns for typical markers and not just
the leukemia/lymphoma ones. Usually only the large hospital or private
IHC labs do these types of panels, so even in the clinical arena this
type of experience isn't easy to get. The IHC techs aren' t the ones
interpreting these results, nor are they ordering the panels. At best
they run patient samples with known positive controls (mostly tonsil),
and order and work up new antibodies to run in these panels--which are
then evaluated by a pathologist for applicability. How is that
different from any other marker run in an IHC lab?
Regarding the need for ASCP-certified techs in the research environment,
I agree with Tim that it should not necessarily be a requirement. I know
that an ASCP-certified tech has general knowledge of histotechnology and
has demonstrated skill with a microtome and staining techiques, and has
the certification to prove it. But that alone does not make them any
more qualified to work in the lab than someone with a BS or MS and
histotechnology experience but no certification. Please don't think I'm
making the case for random hiring of non-certified techs. I believe
firmly in certifications for personal and professional growth. I just
don't think the research environment should base their job requirements
on it. My employment ad usually indicates the person we are looking for
be ASCP certified, or certification eligible. In the absence of the
certification, meeting minimum educational requirements is usually
acceptable.
Finally, I have not paid my dues to ASCP for a very long time and have
no plans to start doing so any time soon. I will eagerly hand over my
money to the NSH because they offer workshops and a publication that are
geared towards the profession. Even the Advance magazine runs more
articles on Histology than the ASCP journal ever did. Perhaps that has
changed, but somehow I doubt it. I too would be steamed if I had paid
all this time and got no value for my $$$s.
Teri Johnson
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, MO 64133
------------------------------
Message: 12
Date: Wed, 16 Nov 2005 17:50:15 -0800
From: "HCS" <int09018 <@t> alphahunt.com>
Subject: [Histonet] Help- triphenyltetrazolium chloride instructions
for staining needed
To: "Histonet" <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <000601c5eb19$4d4b7df0$6601a8c0 <@t> hp>
Content-Type: text/plain; charset="iso-8859-1"
Hi, I am still looking for a reference or procedure for doing the TTC stain
(triphenyltetrazolium chloride). If you might have a procedure I would be
able to use that would be very helpful.
Thanks
LeRoy Brown
HCS
.
------------------------------
Message: 13
Date: Wed, 16 Nov 2005 21:51:35 EST
From: JUDIEWHIT <@t> aol.com
Subject: [Histonet] Re: Histonet Digest, Vol 24, Issue 23
To: histonet <@t> lists.utsouthwestern.edu
Message-ID: <217.e6894da.30ad4a37 <@t> aol.com>
Content-Type: text/plain; charset="US-ASCII"
Hi Jill,
Looks like you are getting ready to open your new lab.
Congrats girlfriend I knew you could do it!
I know some of the techs in Phoenix if you need references let me know.
I am getting ready to come out of retirement and go traveling...looks like I
will be working in a lab in new Mexico for 13 weeks soon, I think the travel
thing will be fun and I can move on to another Histo job in different parts
of
the country.
The very best to you my friend...
Judie W.
------------------------------
Message: 14
Date: Wed, 16 Nov 2005 23:12:28 -0500
From: "Ron Martin" <pathrm35 <@t> adelphia.net>
Subject: [Histonet] biohazard waste removal companies
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <001001c5eb2d$2100bf80$27233418 <@t> Pathrm35>
Content-Type: text/plain; charset="iso-8859-1"
Does any one know of any companies that service southeast Florida for
biohazard waste and chemical waste removal? I am currently using
Environmental Enterprises out of Orlando but lately this company has been
unable to service our needs.
Thanks in advance.
Ron Martin
------------------------------
Message: 15
Date: Thu, 17 Nov 2005 09:05:55 +0100
From: "C.M. van der Loos" <c.m.vanderloos <@t> amc.uva.nl>
Subject: [Histonet] RE: aqueous coverslipping medium
To: histonet <@t> lists.utsouthwestern.edu
Cc: PMonfils <@t> Lifespan.org
Message-ID: <1b34f811b341d2.1b341d21b34f81 <@t> amc.uva.nl>
Content-Type: text/plain; charset="us-ascii"
Paul,
The media you call the "liquid coverslip" is in fact a kind of plastic
polymer that can be mounted up organically after polymerisation. I
experienced that without this additional organic mounting the plastic
polymer will fall off after some years. If this happens you can remove
the plastic completely by soaking the slide in luke warm water.
Then "liquid-coverslip" again.
Although most of those products state in their data sheet that it
should be used without a coverslip, it also works fine WITH a
coverslip. The only thing you should not do is harden at 60-70C as you
normally do (it causes air bubbles!). Just leave the slides at room
temp.
Chris van der Loos, PhD
Dept. of Pathology
Academic Medical Center M2-230
Meibergdreef 9
NL-1105 AZ Amsterdam
The Netherlands
Date: Wed, 16 Nov 2005 12:40:06 -0500
From: "Monfils, Paul" <PMonfils <@t> Lifespan.org>
Subject: [Histonet] aqueous coverslipping medium
To: "'histonet <@t> lists.utsouthwestern.edu'"
What brand do you prefer for aqueous mounting medium to be used with a
coverslip? I have several good hydrocarbon-based coverslipping media,
and
also some good aqueous media to be used without coverslips (so-called
liquid
coverslip), but I don't have an aqueous coverslipping medium I am
completely
satisfied with. Applications would include immunofluorescence and a
few
enzyme stains, as well as lipid stains and other techniques that are
not
compatible with hydrocarbon-based media. Are there any such media
that
harden after application, to produce a permanent slide?
------------------------------
Message: 16
Date: Thu, 17 Nov 2005 11:31:40 +0200
From: louise renton <louise.renton <@t> gmail.com>
Subject: [Histonet] hard tissue distortion
To: Histonet <@t> lists.utsouthwestern.edu
Message-ID:
<e483362e0511170131v48526345h12c4c692c694d2b0 <@t> mail.gmail.com>
Content-Type: text/plain; charset=ISO-8859-1
Helo all,
I've been asked this question by a postgrad and I wasn't able to
answer it to my satisfaction. The question was - how much distortion
occurs in tissue (in this case teeth) that have been fixed in 70%
alcohol, processed by hand and embedded in MMA?, Sections were cut at
6um, stained free floating and mounted in Entellan on glass slides.
Any thoughts/references? I would be most grateful for anything The
post grad is doing histometry on healing furcation defects, and is
wondering if there are any variables that he needs to mentionor take
into consideration.
Best regards
--
Louise Renton
Bone Research Unit
University of the Witwatersrand
Johannesburg
South Africa
"....onwards through the fog!"
------------------------------
Message: 17
Date: Thu, 17 Nov 2005 06:10:19 -0800 (PST)
From: Histology SLU <sluhisto <@t> yahoo.com>
Subject: [Histonet] CD31 staining histiocytes???
To: "histonet <@t> lists.utsouthwestern.edu"
<histonet <@t> lists.utsouthwestern.edu>
Message-ID: <20051117141019.29359.qmail <@t> web51003.mail.yahoo.com>
Content-Type: text/plain; charset=iso-8859-1
Hello all:
Is anyone out there using Dako CD31 and getting histiocyte staining in
human skin??? Our dermpath is seeing this and is questioning it. We are
using it at 1:100 with the biocare pressure cooker with Biocare Diva
decloaker solution. Our detection system is Envision +. Any thoughts???
Susan
Histopathology Supervisor
St. Louis University Medical School
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------------------------------
Message: 18
Date: Thu, 17 Nov 2005 09:52:03 -0500
From: Angela McNabola <angela.mcnabola.b <@t> bayer.com>
Subject: [Histonet] CD31 Santa Cruz update
To: histonet <@t> lists.utsouthwestern.edu
Message-ID:
<OFDC366556.4DBD8939-ON852570BC.0051AB43-852570BC.0051AB71 <@t> bayer.com>
Content-Type: text/plain; charset=US-ASCII
Hello all,
I received a new lot of CD31 yesterday, provided to me by Santa Cruz. I
have
not yet had time to try it out (FFPE-mouse xenografts), but I have been told
that it is working.
I know that several of you have been waiting on this, so now might be a good
time to give them a call.
Angela
Angela McNabola MS, HT(ASCP)SLS, QIHC
Scientist
Bayer Healthcare
400 Morgan Lane
West Haven, CT 06516
203-812-5001
fax# 203-812-5820
angela.mcnabola.b <@t> bayer.com
------------------------------
Message: 19
Date: Thu, 17 Nov 2005 14:56:02 -0000
From: "Edwards, R.E." <ree3 <@t> leicester.ac.uk>
Subject: RE: [Histonet] E: beta-2- microglobulin
To: <Histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<DC88BEDFD1FC3F468D0376A7C75465F70BD8EF7F <@t> Saffron.cfs.le.ac.uk>
Content-Type: text/plain; charset="iso-8859-1"
Looking for an antibody to the above that works, as ever, on
paraffin processed mouse tissues...
Many thanks
Richard Edwards
MRC TOX UNIT
LEICESTER...U.K....
------------------------------
Message: 20
Date: Thu, 17 Nov 2005 10:36:34 -0500
From: "Kinsley, David" <david.kinsley <@t> spcorp.com>
Subject: [Histonet] fixed jaw clamps/ large size specimens
To: "'histonet <@t> lists.utsouthwestern.edu'"
<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<79A75F7AD4BFAE4CA27C8AABE3F75D9A0E6C42 <@t> KENMSG22.us.schp.com>
Content-Type: text/plain
I'm looking to section some larger size specimens, particularly rat hind paw
samples and am looking for cassettes that are slightly larger than the mega
cassette but not as large as the super mega cassette. (Approximately 2"
long by 1.5" wide by 1/2 to 3/4" deep) I have called some vendors and have
had little luck.
The only suggestion was to purchase a fixed jaw clamp for my microtome
(Microm HM355) and try to use that with the super mega cassette.
Does anyone have any experience with these types of specimens? Any
suggestions on cassettes or microtome adapters?
thanks
David Kinsley
Schering-Plough Research Institute
Kenilworth, NJ 07033
908-740-4669
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This message and any attachments are solely for the intended recipient. If
you are not the intended recipient, disclosure, copying, use or distribution
of the information included in this message is prohibited -- Please
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------------------------------
Message: 21
Date: Thu, 17 Nov 2005 10:09:09 -0600
From: "Bauer, Karen" <Bauer.Karen <@t> mayo.edu>
Subject: [Histonet] Dissecting Microscopes
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<FAEEA161EC98C6439D4892E161BCE18A1F230C <@t> LMMAILVS1.ad.lmmhs.org>
Content-Type: text/plain; charset="iso-8859-1"
Good morning! I need to purchase a new dissecting microscope for our
pathologists. Ours is getting old and needs to be updated. We mainly use
it for finding glomeruli in kidney biopsies to make sure there is adequate
tissue for certain types of testing. I've looked online and have requested
some quotes, but I'm curious to know what others are using. Any
suggestions?
As always, thank you.
Karen L. Bauer HT(ASCP)
Histology Supervisor
Luther Hospital
Eau Claire, WI
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Message: 22
Date: Thu, 17 Nov 2005 09:31:15 -0800 (PST)
From: Rene J Buesa <rjbuesa <@t> yahoo.com>
Subject: Re: [Histonet] fixed jaw clamps/ large size specimens
To: "Kinsley, David" <david.kinsley <@t> spcorp.com>,
"'histonet <@t> lists.utsouthwestern.edu'"
<histonet <@t> lists.utsouthwestern.edu>
Message-ID: <20051117173115.47213.qmail <@t> web61215.mail.yahoo.com>
Content-Type: text/plain; charset=iso-8859-1
David:
In 1988 we began cutting whole prostate slices and we could not find
neither megacassettes nor molds, so we made ours.
For cassettes I used small round plastic containers, cut to an appropriate
height with drilled holes and stappled 2 together, with the specimen in
between. They work perfectly.
As molds I cut pieces of wood of the required size and used aluminum
sheets bought in a hardware to "wrap" around the wood piece and made them
"melted paraffin leak proff". They also worked beautifully and, even when at
last we bought the available cassettes and molds, for really large specimens
(up to 3" x 4" ) we still use them.
For those large paraffin blocks I used the very very old technique of
adhering them to small 1"x1"x 2" blocks of wood, using a hot spatula, to be
clamped to the microtome.
Hope this "barrage" of "oldies" will help!
Rene J.
"Kinsley, David" <david.kinsley <@t> spcorp.com> wrote:
I'm looking to section some larger size specimens, particularly rat hind
paw
samples and am looking for cassettes that are slightly larger than the mega
cassette but not as large as the super mega cassette. (Approximately 2"
long by 1.5" wide by 1/2 to 3/4" deep) I have called some vendors and have
had little luck.
The only suggestion was to purchase a fixed jaw clamp for my microtome
(Microm HM355) and try to use that with the super mega cassette.
Does anyone have any experience with these types of specimens? Any
suggestions on cassettes or microtome adapters?
thanks
David Kinsley
Schering-Plough Research Institute
Kenilworth, NJ 07033
908-740-4669
*********************************************************************
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you are not the intended recipient, disclosure, copying, use or distribution
of the information included in this message is prohibited -- Please
immediately and permanently delete.
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