[Histonet] RE: Histonet Digest, Vol 4, Issue 23
Dorothy.L.Webb <@t> HealthPartners.Com
Dorothy.L.Webb <@t> HealthPartners.Com
Wed Mar 24 14:22:48 CST 2004
You can postfix smears with formalin fumes by using a coplin jar with
formalin soaked gauze in the cover for 20 minutes.
Or, you can fix the smears in equal parts of hydrogen peroxide and methyl
alcohol for 15 minutes. These both help to have the cells adhere to the
slide. Hope this helps! Dorothy Webb, Regions Hospital, St. Paul, MN.
-----Original Message-----
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Sent: Friday, March 19, 2004 12:00 PM
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Subject: Histonet Digest, Vol 4, Issue 23
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Today's Topics:
1. NCSHT Spring Meeting (Delorise Williams)
2. need help with IHC detection of human cells in mouse tissue
(Garlits, John)
3. (no subject) (Yang Wang)
4. Extra tissues in LCM capture (Yang Wang)
5. RE: Iron stain on frozen sections (Tony Henwood)
6. RE: Placental section ihc... (Rena Fail)
7. Using H & E slides on the Benchmark XT (Joe Nocito)
8. DAB-Mn histochemistry for superoxide: help to remove
background (Subratab)
9. "Animal" person seeking clinical folks help! (Angela McNabola)
10. Re: "Animal" person seeking clinical folks help! (NICK KIRK)
----------------------------------------------------------------------
Message: 1
Date: Thu, 18 Mar 2004 18:46:08 -0500
From: "Delorise Williams" <DWilliams <@t> ciit.org>
Subject: [Histonet] NCSHT Spring Meeting
To: <histonet <@t> pathology.swmed.edu>
Cc: Delorise Williams <DWilliams <@t> ciit.org>
Message-ID: <12816CB59E68184F9F5F28063E68B047285DC0 <@t> xsrvr.ciit.org>
Content-Type: text/plain; charset="iso-8859-1"
The North Carolina Society OF Histopathology Technologists presents the 2004
Spring Meeting on April 23 &24 at the Hilton Wilmington Riverside in
Wilmington NC. Below is a summary of the program:
Friday: April 23, 2004
Using Histological Techniques for Protein and mRNA
Expression Analysis in Pharmaceutical Research and 8:15-9:15AM Drug
Discovery
Stephanie Broka, BS
GlaxoSmithKline, Research Triangle Park, NC
9:15 - 10:15 AM Basic Immunology and Immunohistochemistry
Godfrey Guerzon , MS,MBA, HT, DLM (ASCP)
New Hanover Regional Medical
Center,Wilmington, NC
11:00 - 12:00 Organize for Success
Beth Moore,MT (ASCP) DLM, MBA
New Hanover Regional Medical Center,
Wilmington, NC
WORKSHOP # 1 (1:30-4:30)
HT (ASCP) Examination Readiness-The Practical Exam-Robert Lott, HTL(ASCP)
Baptist Health System,Birmingham, Al
WORKSHOP # 2 (1:30-4:30)
Preparation of Tissue for Evaluation of Sexually Dimorphic Nuclei in Rat
Brain-Melanie Struve, BS and Renee Thacker,BS, HT(ASCP)
CIIT Centers for Health Research, Research Triangle Park, NC
Saturday: April 24,2004
WORKSHOP # 3 (8:15-12:00)
HT (ASCP) Examination Readiness-The Written Exam-Robert Lott, HTL (ASCP)
Baptist Health Systems, Birmingham, Al
WORKSHOP #4 (8:15-12:00)
Catalyzed Signal Amplification system-Leslie Sells, BS, HTL, Technical
Support Representative, DakoCytomation
WORKSHOP # 5 (1:00-4:30)
Dyes and Tissue: Chemistry Mechanism of Staining-Bert Dotson,MBA, HTL (ASCP)
Duke University Medical Center, Durham, NC
WORKSHOP # 6 (1:00-4:30)
Microwave: Routine Histological Application-Theory to Practice
Jim Milios, International Applications Specialist, Milestone Srl., Italy
Delorise Williams
CIIT Centers for Health Research
PO Box 12137
Research Triangle Park, NC 27709
(919) 558-1200
Voice Mail-(919) 558-1252
Fax-(919) 558-1300
------------------------------
Message: 2
Date: Thu, 18 Mar 2004 17:51:40 -0600
From: "Garlits, John" <John.Garlits <@t> stjude.org>
Subject: [Histonet] need help with IHC detection of human cells in
mouse tissue
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<1E0CC447E59C974CA5C7160D2A2854EC238ABF <@t> SJMEMXMB04.stjude.sjcrh.local>
Content-Type: text/plain; charset="iso-8859-1"
Hi,
I was wondering if anybody could help me out. We want to look for human
cells engrafted in mice, particularly in formalin-fixed, acid decalcified,
paraffin-embedded tissue. I have so far tried two anti-human beta-2
microglobulin antibodies.
The first I tried has been used in human/sheep transplant tissue, and I
tried it, but unfortunately it is an IgM kappa, so it failed with the
Biogenex mouse-on-mouse IHC kit, which it turns out is not made for IgM
antibodies. If anybody is aware of a good way to use a mouse IgM on both
human and mouse tissue, I'd love to know. I have looked for a good
secondary antibody for this purpose, but have had no luck so far.
The second antibody I tried is from Novocastra and distributed by Vector
Labs. It is a rabbit polyclonal to human beta-2-microglobulin. The trouble
I had was nonspecific binding in control mouse tissue. I was thinking to
use some of the Biogenex mouse-on-mouse kit reagents to help block mouse
antigens, but I do not know if this would actually work. The second problem
with this antibody was that I did not always see positive marking in human
tissue (especially osteocytes), which I thought should be pretty well all
positive since it is human tissue. Is the expression of
beta-2-microglobulin so variable?
Has anyone tried any other general human cell marker in other animals? I
did a quick search for beta actin, but it seems most of those cross-react
with mouse.
Thank you!
John Garlits, M.S.
Senior Research Technician
Hematology Oncology Division
Experimental Hematology Department
St. Jude Children's Research Hospital
332 N Lauderdale
Memphis, TN 38108
------------------------------
Message: 3
Date: Thu, 18 Mar 2004 18:53:28 -0500
From: Yang Wang <Yang.Wang <@t> tufts.edu>
Subject: [Histonet] (no subject)
To: "histonet <@t> lists.utsouthwestern.edu"
<histonet <@t> lists.utsouthwestern.edu>
Message-ID: <1079654008.405a367856f5c <@t> webmail.tufts.edu>
Content-Type: text/plain; charset=ISO-8859-1
Hi, dear histonet friends:
We need help! We have been working on the conditions for LCM for about 7
months, which is still very frustrating:-(.We tried to capture single cells
from intestinal tissues. However, the major problem we are facing now is
that
we always get extra tissues from the capture. (We could pull off a whole
villi
when we capture a single cellL) .We used the standard protocols provided
by "ARCTURUS" for the tissue section and dehydration.
Here is what we have done:
1.We cut 5um frozen sections and do either "pre" or "post" fixation in 4%
paraformaldehyde.
a. "Pre": fix 2h on ice before frozen, since we are working on GFP
expressing
tissue, which required pre-fixation.
b. " Post": fix 15-30 min after frozen
2.The slides will then be dehydrated by: 75% ethyl 30s, 95% ethy 30s, 100%
ethyl 1min and xylene 5min, then air dry 30min.
We always pull off extra tissues, which adjacent to the cell interested. We
tried treat slides and cap with "ARCTURUS prep strip". But it didn't help.
We
are wondering whether we need specific treatment with slides (we use fisher
superfrost plus)? Or our procedures are not proper?
Thanks a lot for help!
Yang Wang
New England Medical Center
Yang.wang <@t> tufts.edu
------------------------------
Message: 4
Date: Thu, 18 Mar 2004 18:56:48 -0500
From: Yang Wang <Yang.Wang <@t> tufts.edu>
Subject: [Histonet] Extra tissues in LCM capture
To: "histonet <@t> lists.utsouthwestern.edu"
<histonet <@t> lists.utsouthwestern.edu>
Cc: Yang.Wang <@t> tufts.edu
Message-ID: <1079654208.405a3740c18b2 <@t> webmail.tufts.edu>
Content-Type: text/plain; charset=ISO-8859-1
Hi, dear histonet friends:
We need help! We have been working on the conditions for LCM for about 7
months, which is still very frustrating:-(.We tried to capture single cells
from intestinal tissues. However, the major problem we are facing now is
that
we always get extra tissues from the capture. (We could pull off a whole
villi
when we capture a single cellL) .We used the standard protocols provided
by "ARCTURUS" for the tissue section and dehydration.
Here is what we have done:
1.We cut 5um frozen sections and do either "pre" or "post" fixation in 4%
paraformaldehyde.
a. "Pre": fix 2h on ice before frozen, since we are working on GFP
expressing
tissue, which required pre-fixation.
b. " Post": fix 15-30 min after frozen
2.The slides will then be dehydrated by: 75% ethyl 30s, 95% ethy 30s, 100%
ethyl 1min and xylene 5min, then air dry 30min.
We always pull off extra tissues, which adjacent to the cell interested. We
tried to treat slides and cap with "ARCTURUS prep strip". But it didn't
help.
We are wondering whether we need specific treatment with slides (we use
fisher
superfrost plus)? Or our procedures are not proper?
Thanks a lot for help!
Yang Wang
New England Medical Center
Yang.wang <@t> tufts.edu
------------------------------
Message: 5
Date: Fri, 19 Mar 2004 11:38:27 +1100
From: Tony Henwood <AnthonyH <@t> chw.edu.au>
Subject: RE: [Histonet] Iron stain on frozen sections
To: "'Connolly, Brett M'" <brett_connolly <@t> merck.com>, "'HISTONET'
(histonet <@t> lists.utsouthwestern.edu)"
<histonet <@t> lists.utsouthwestern.edu>
Message-ID: <1CF2E2E5BB36D5119E7A0008C791F3740800E167 <@t> simba.kids>
Content-Type: text/plain; charset="iso-8859-1"
Brett,
Following is an excerpt from a paper I published a year or so ago:
Henwood, A.F., (2002) "Microwave Perl's Stain for urgent frozen sections"
Aust J Med Sc 23(2):68-69).
Tony Henwood JP, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager
The Children's Hospital at Westmead,
Locked Bag 4001, Westmead, 2145, AUSTRALIA.
Tel: 612 9845 3306
Fax: 612 9845 3318
http://www.histosearch.com/homepages/TonyHenwood/default.html
http://us.geocities.com/tonyhenwoodau/index.html
MICROWAVE PERLS' STAIN FOR URGENT FROZEN SECTIONS
Anthony F. Henwood
Laboratory Manager,
Histopathology,
The Children's Hospital at Westmead,
Locked Bag 4001,
Westmead, 2145, AUSTRALIA
Abstract
A rapid Perls' Stain for hemosiderin using a microwave oven is described. It
takes one minute to perform and is particularly suitable for urgent frozen
section diagnosis.
Introduction
Urgent frozen section diagnosis is usually restricted to the interpretation
of haematoxylin and eosin stained sections (1). With the increased
utilisation of microwave modifications of routine special stains, it is
possible to perform a range of such stains on frozen sections (1). To be
applicable to urgent frozen section diagnosis, a special stain needs to be
rapid, preferably taking 1-2 minutes to perform, easy to prepare, using
stock solutions with few preparation steps and produce results equal to
those obtained on paraffin sections.
The following microwave Perls' technique for hemosiderin takes about one
minute to perform.
Method
Frozen sections can be fixed in Shoobridge's fixative (10ml concentrated
formalin in 30ml absolute alcohol) (2) or 95% alcohol. Kennedy and Forbes
(1) have also successfully used Wolman's fixative (5% acetic acid in
absolute ethanol).
1. Rinse fixed frozen sections in water and then place in a coplin jar
containing 20m1 each of 2% Potassium Ferrocyanide and 2% Hydrochloric acid.
Place in the microwave oven and microwave for 20 seconds at 650 watts.
Slides can be left in this solution for up to 3 minutes. Remove slides if
the solution begins to turn cloudy.
2. Rinse slides in water.
3. Counterstain using agitation, with eosin (as routinely used for the
frozen section HE) for 5-7 seconds.
Other counterstains such as Nuclear Fast Red (3) or 0.25% basic fuchsin (4)
can also be used, though eosin is readily available at the frozen section
site and works quite well.
Since the frozen block is usually fixed and processed to paraffin, sections
of this block should also be stained with the routine Perls' as a quality
control procedure.
Discussion
Iron in the body is stored in the forms of hemosiderin (ferric hydroxide
polymer) or ferritin (a ferrous iron-protein complex) (5). Iron in tissues
occurs mainly in the ferric state (6,7).
Microscopically, hemosiderin appears similar to other yellow to brown
pigments, such as melanin and fine carbon dust. Macrophages can contain any
of these pigments. Heavily pigmented macrophages, often masking the cell
nucleus, can be confused with malignant melanoma. The identification of the
pigment as being hemosiderin, especially at the time of frozen section, is
diagnostically useful.
The rapid Perls' stain described above uses the same solution as used by
Schaffner (4). This solution is easier to prepare than others reported in
the literature (3). The stain takes approximately one minute to perform and
gives results equivalent to those obtained in paraffin sections. It is
especially applicable to frozen section diagnosis.
References:
1. Kennedy, A., Foulis, A.K. (1989) "Use of microwave oven improves
morphological and staining of cryostat sections", J.Clin.Pathol. 42:101-105.
2. Shoobridge, M.P.K., (1978) "Improving frozen sections by wet fixation",
(Abstract) Pathology 10:195.
3. Brian, N.T., (1983) "Rapid Metallic Histological staining using the
microwave oven", J.Histotechnol. 6(3): 125-129.
4. Schaffner, R., (1986) "The Perls' Iron staining procedure for use in the
microwave oven using a temperature probe", J.Histotechnol. 9(2): 107-108.
5. Barka, T., Anderson, P.J., (1963) "Histochemistry: Theory, practice and
bibliography" Harper & Row Publishers Inc, New York, p172-174.
6. Davenport, H.A., (1961) "Histological and Histochemical Technics" W.B.
Saunders Co., Philadelphia, 280-284.
7. Gabe, M., (1976) "Histological Techniques" Masson, Paris, p311-317.
-----Original Message-----
From: Connolly, Brett M [mailto:brett_connolly <@t> merck.com]
Sent: Friday, 19 March 2004 6:03 AM
To: 'HISTONET' (histonet <@t> lists.utsouthwestern.edu)
Subject: [Histonet] Iron stain on frozen sections
Along with the bone marrow thread.....
Has anyone had experience with iron stains on frozen sections? What
fixative? Procedure?
Thx,
Brett
Brett M. Connolly, Ph.D.
Merck & Co., Inc.
MRL, Imaging Research
WP26A-3000
PO Box 4
West Point, PA 19486
PH 215-652-2501
fax. 215-652-2075
e-mail. brett_connolly <@t> merck.com
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Message: 6
Date: Thu, 18 Mar 2004 22:09:52 -0500
From: Rena Fail <RFail <@t> Charleston.net>
Subject: RE: [Histonet] Placental section ihc...
To: nyilmaz <@t> mersin.edu.tr, histonet <@t> lists.utsouthwestern.edu
Message-ID: <010501c40d5f$cde15f50$dc10a6a5 <@t> rena>
Content-Type: text/plain; charset=us-ascii
We are on occasion asked to stain placenta tissue for IHC. We use
non-fat milk as a protein block. No problems with background
Rena Fail
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of
nyilmaz <@t> mersin.edu.tr
Sent: Thursday, March 18, 2004 3:29 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Placental section ihc...
We are using placental tissue for immunohistochemistry, and have some
problems about background staining especially with serum remnants in the
vessels. Does anybody have ideas about this problem. If you inform us
we'd be greatly appreciate...
Nejat Yilmaz PhD
Mersin University Medical School
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------------------------------
Message: 7
Date: Fri, 19 Mar 2004 06:51:05 -0600
From: "Joe Nocito" <JNocito <@t> Pathreflab.com>
Subject: [Histonet] Using H & E slides on the Benchmark XT
To: "Histonet" <histonet <@t> pathology.swmed.edu>
Message-ID: <JFEMICGBHEGPLAMIJPJPIEMJCCAA.JNocito <@t> Pathreflab.com>
Content-Type: text/plain; charset="iso-8859-1"
Morning Histoland,
We have acquired the Ventana Benchmark XT and I really need some
help. When
we were performing the slides manually, we were able to take an H&E slide,
take off the coverslip and run an immuno on it. The doctors fell in love
with this since we processes a lot of cervical biopsies and sometimes the
lesion is not there on recuts. We have had a problem with H&E slides not
picking up the immuno staining. I set up different programs using the wet
slide protocol, but I'm having problems still.
Would anyone be willing to share their protocol with me?
Thanks
Joe Nocito, BS, HT (ASCP) QIHC
Histology Manager
Pathology Reference Lab
San Antonio, TX 78205
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Message: 8
Date: Fri, 19 Mar 2004 20:20:32 +0600
From: Subratab <subratab <@t> bdonline.com>
Subject: [Histonet] DAB-Mn histochemistry for superoxide: help to
remove background
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <200403191425.i2JEPrGC023459 <@t> mailout.proshikanet.com>
Content-Type: text/plain; charset="iso-8859-1";
Dear histo-experts
I am trying to stain superoxide producing cells on frozen tissue sections of
rat kidney. I am using Brigg's (and Karnovsky) cytochemical method in
unfixed tissue. The reaction medium contains 1 mg/ml DAB, 1 mM sodium azide
and 0.5 mM MnCl2 in 100mM Tris-HCl (pH 7.2). Incubation time 30 minutes at
37 degree C. (The principle of the reaction is that intracellular superoxide
will oxidize Mn++ to Mn+++; then Mn+++ will in turn cause DAB oxidation and
precipitaion).
MY PROBLEM: I am getting profuse DAB staining throughout the
tubulo-interstitial area (glomerulus is clean). Most probably its from
endogenous peroxidase activity. So I tried to block endo perox by H2O2 (and
also by sodium azide, and H2O2+azide) before incubation with reaction
medium. This way I have removed tubulo-interstitial DAB staining. But I am
not getting any positive stain. I suspect that my blocking step is somehow
damaging (blocking) superoxide producing enzymes as well.
At this stage I need some expert opinion. How can I specifically block
endogenous peroxidase keeping other enzymes intact? Hope to get some
effective ways from histo-experts.
Thanks in advance
Subrata Biswas MD
Lab de Fisiopatologia Renal
Uni of Campinas, SP, Brazil.
------------------------------
Message: 9
Date: Fri, 19 Mar 2004 09:31:13 -0500
From: Angela McNabola <angela.mcnabola.b <@t> bayer.com>
Subject: [Histonet] "Animal" person seeking clinical folks help!
To: histonet <@t> lists.utsouthwestern.edu
Cc: Maura Broggi <maura.broggi.b <@t> bayer.com>
Message-ID:
<OFD9CBEDCA.7EBE79E7-ON85256E5C.004EC207-85256E5C.004FC344 <@t> bayer.com>
Content-Type: text/plain; charset=US-ASCII
Hi all,
I soliciting protocols that any of you may be willing to share on how to
make
slides suitable for IHC using FNA's. I am looking for how to best make
slide
smears (I guess!). Clinical sites will be collecting samples from patients
and
sending the slides to my lab for staining/evaluation.
We are fairly new to this, even new to processing human samples, so any help
you
can provide would be greatly appreciated. Also, keep in mind that we
potentially may be receiving samples from all over the world, so the easier
the
better since we will be asking seeral sites to do it all the same way
thanks in advance!
Angela McNabola, MS, HT(ACSP)QIHC, SLS, RLATG
Bayer Healthcare
400 Morgan Lane
West Haven, CT 06516
angela.mcnabola.b <@t> bayer.com
------------------------------
Message: 10
Date: Fri, 19 Mar 2004 15:25:09 +0000 (GMT)
From: NICK KIRK <nick.kirk3 <@t> btopenworld.com>
Subject: Re: [Histonet] "Animal" person seeking clinical folks help!
To: Angela McNabola <angela.mcnabola.b <@t> bayer.com>, Histonet
<histonet <@t> lists.utsouthwestern.edu>
Message-ID: <20040319152509.87494.qmail <@t> web86308.mail.ukl.yahoo.com>
Content-Type: text/plain; charset=iso-8859-1
Angela
Here's a useful recipe/technique for cytological material. The immuno
results are very good, plus you have the bonus of being able to keep any
residual material for a longer period.
RECIPE for PEG/IMS fixative
3% Polyethylene glycol in 50% Industrial methylated spirit (IMS) {=99%
ethanol}.
---------------------------------
Method
1. Spin cells down
2. Perform blood lysis (if required) with Ammonium chloride solution
3. Re-suspend in PEG/IMS fixative
4. Leave to fix for 2 hours
5. Make cytospins
6. Air dry completely
7. Place in 95% I.M.S. for at least 10 minutes to remove PEG
8. Immunostain
Any left over material can be stored in the PEG/IMS fixative for prolonged
periods of time.
Hope this is of some help
Nick Kirk
Histopathology
Hinchingbrooke Hospital
Huntingdon
England
Angela McNabola <angela.mcnabola.b <@t> bayer.com> wrote:
Hi all,
I soliciting protocols that any of you may be willing to share on how to
make
slides suitable for IHC using FNA's. I am looking for how to best make slide
smears (I guess!). Clinical sites will be collecting samples from patients
and
sending the slides to my lab for staining/evaluation.
We are fairly new to this, even new to processing human samples, so any help
you
can provide would be greatly appreciated. Also, keep in mind that we
potentially may be receiving samples from all over the world, so the easier
the
better since we will be asking seeral sites to do it all the same way
thanks in advance!
Angela McNabola, MS, HT(ACSP)QIHC, SLS, RLATG
Bayer Healthcare
400 Morgan Lane
West Haven, CT 06516
angela.mcnabola.b <@t> bayer.com
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End of Histonet Digest, Vol 4, Issue 23
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