[Histonet] RE: Millers Elastin Stain HELP
White, Lori
lwhite <@t> lakeridgehealth.on.ca
Tue Feb 1 12:56:24 CST 2005
-----Original Message-----
Julia,
Inter-medico in Canada has just released a kit stain for Millers.
Lori W
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Subject: Histonet Digest, Vol 15, Issue 2
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Today's Topics:
1. RE: aqueous mounting medium (Amanda MacFarlane)
2. RE: Excessive shrinkage[Scanned] (Gayle Callis)
3. Re: Mouse liver fixation (Gayle Callis)
4. p34 (Richard Cartun)
5. Professional Qualifications in UK (Prior, Andrew)
6. Re: Millers Elastin Stain HELP (Prior, Andrew)
7. RE: yeast and bacteria on slides (Bonner, Janet)
8. Re: Formalin pigment (John Kiernan)
9. another background / non-specific staining problem (Subratab)
10. RE: HRP-conjugated secondary and biotinylated-secondary (Subratab)
----------------------------------------------------------------------
Message: 1
Date: Tue, 1 Feb 2005 10:20:38 -0500 (EST)
From: "Amanda MacFarlane" <am458 <@t> cornell.edu>
Subject: [Histonet] RE: aqueous mounting medium
To: "C.M. van der Loos" <c.m.vanderloos <@t> amc.uva.nl>
Cc: histonet <@t> lists.utsouthwestern.edu
Message-ID: <1935.128.253.96.73.1107271238.squirrel <@t> 128.253.96.73>
Content-Type: text/plain;charset=iso-8859-1
This is good to know. I was under the impression from what I've read
that
the chromogenic product of beta-gal was soluble in xylene, thus the need
for aqueous mounting medium.
Thanks,
Amanda
>
> Hi Amanda,
> From my experiments with X-gal as chromogen for IHC techniques it
appeared
> that the blue-greenish reaction product (with X-gal +
ferro/ferricyanide)
> is very stable and survives any mounting medium. You may dehydrate and
> mount organically.
> Chris van der Loos, PhD
> Dept. of Pathology
> Academical Medical Center M2-230
> Meibergdreef 9
> NL-1105 AZ Amsterdam
> The Netherlands
> phone: +31 20 5665631
> fax: +31 20 6960389
> e-mail: c.m.vanderloos <@t> amc.uva.nl
>
> ----- Original Message ----- From "Amanda MacFarlane" Date Mon,
31
> Jan 2005 11:38:24 -0500 (EST) To Histonet <@t> lists.utsouthwestern.edu
> Subject [Histonet] aqueous mounting medium
> I have stained whole tissues for beta-galactosidase (X-gal) and am now
> cryosectioning the tissues. I have been fixing the sections in 4% PFA
> overnight at 4 degrees Celsius and cover slipping with 70% glycerol.
> Coverslipping with glycerol is a time-consuming pain in the butt so
I've
> checked out glycerol vinyl alcohol aqueous (GVA) mounting solution
(Zymed)
> and Supermount aqueous mounting medium (Biogenex) as alternatives. It
> appears that these products have not been tested with beta-gal
staining,
> so I'm wondering if people use these products and/or what people
> recommend. Thanks.
>
>
> Amanda MacFarlane, PhD
> Division of Nutritional Sciences
> Cornell University
>
Amanda MacFarlane
Division of Nutritional Sciences
Cornell University
------------------------------
Message: 2
Date: Tue, 01 Feb 2005 08:20:47 -0700
From: Gayle Callis <gcallis <@t> montana.edu>
Subject: RE: [Histonet] Excessive shrinkage[Scanned]
To: Kemlo Rogerson <Kemlo.Rogerson <@t> elht.nhs.uk>,
Histonet <@t> lists.utsouthwestern.edu
Message-ID:
<6.0.0.22.1.20050201081406.01b4a6f8 <@t> gemini.msu.montana.edu>
Content-Type: text/plain; charset="us-ascii"; format=flowed
Kemlo,
Bouins uses:
saturated picric acid, aqueous
formaldehyde
acetic acid
and you are correct about removing picric acid, some use 70% alcohol
rinses
or 70% alcohol containing lithium carbonate.
8 hours is a common fixation time, with no longer than 72 hours
recommended. I recall it made our tissues crunchier.
Also, test the temperature of your paraffin, has it changed to higher?
All processing creates some hardness, with approx 25% shrinkage in
tissues
due to water removal and heat of paraffin, and is unavoidable. Have an
interesting publication where the group tested the shrinkage of paraffin
processing and plastic processing.
At 01:11 AM 2/1/2005, you wrote:
>If it normally doesn't happen then what makes you think it was the
Bouins
>and is there anything else you changed. Bouins is alcoholic Picric Acid
>isn't it? I assume it's an additive coagulant fixative and if you leave
>tissue in it too long it goes rock hard. Dimly in my memory we used to
wash
>out the picric acid but I don't understand why we put it in, in the
first
>place. Has your processing changed? Are you leaving the tissue in
Bouins too
>long?
>
>-----Original Message-----
>From: Kim O'Sullivan [mailto:Kim.Osullivan <@t> med.monash.edu.au]
>Sent: 01 February 2005 06:41
>To: histonet <@t> lists.utsouthwestern.edu
>Subject: [Histonet] Excessive shrinkage[Scanned]
>
>Hi all,
>
>Can anyone tell me what may cause bouins fixed tissue (in this case
mice
>kidneys's) to shrink after tissue processing (6 hour).This normally
does not
>happen.
>
>ta
>
>Kim
>
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Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)
------------------------------
Message: 3
Date: Tue, 01 Feb 2005 08:32:38 -0700
From: Gayle Callis <gcallis <@t> montana.edu>
Subject: [Histonet] Re: Mouse liver fixation
To: "Andrew MacDuff" <Andrew.MacDuff <@t> ed.ac.uk>,
Histonet <@t> lists.utsouthwestern.edu
Message-ID:
<6.0.0.22.1.20050201082321.01b5fe20 <@t> gemini.msu.montana.edu>
Content-Type: text/plain; charset="us-ascii"; format=flowed
Andrew,
We had terrible problems with mouse liver when whole livers were
immersed
in only 50 ml neutral buffered formalin and left to sit around. The
tissue
brought in a week later was NOT fixed internally, it was still red. If
you can perfuse the animal, dissect out liver and immerse into formalin
1:20 volume. Or fix for a day, take liver out, slice it and return to
fresh formalin.
Liver is very homogenous, dense and surprisingly takes a longer time to
fix
than mouse lung. If liver is dissected out, bread slicing it for
better
fixation is advisable. Slices are embedded cut sides down unless you
want
to embed a whole liver - a rather large chunk of tissue for such a
little
critter. If you plan on a whole liver, be sure to change the fixative
after a day or so to replenish the formalin and this is advisable with
sliced liver too.
Whatever you do, make sure fixative can reach internal parts of liver or
you get some autolyzed tissue.
At 05:12 AM 2/1/2005, you wrote:
>Dear All
>
>Thanks for your great advice last time on fixing mouse lungs. You'll be
>glad to know we've got it working and have some nice H+E slides and the
>immunos will be staring soon.
>Can you help again? My boss has decided to look at the mouse's liver as
>well (poor thing!) and I'd be grateful if you have any tips on fixing
>liver. Can you just remove the liver and place it in a vial of formalin
or
>does it need perfused etc?
>Looking forward to hearing from you all again.
>Regards
>Andrew
>
>Andrew MacDuff
>Clinical Research Fellow
>Wilkie Laboratory
>Medical School
>Edinburgh University
>Teviot Place
>Edinburgh
>andrew.macduff <@t> ed.ac.uk
>_______________________________________________
>Histonet mailing list
>Histonet <@t> lists.utsouthwestern.edu
>http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)
------------------------------
Message: 4
Date: Tue, 01 Feb 2005 10:33:12 -0500
From: "Richard Cartun" <Rcartun <@t> harthosp.org>
Subject: [Histonet] p34
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <s1ff5afe.011 <@t> harthosp.org>
Content-Type: text/plain; charset=US-ASCII
Is anyone doing IHC for p34 on formalin-fixed, paraffin-embedded tissue?
If so, whose antibody are you using? Thank you!
Richard
Richard W. Cartun, Ph.D.
Director, Immunopathology & Histology
Assistant Director, Anatomic Pathology
Hartford Hospital
80 Seymour Street
Hartford, CT 06102
(860) 545-1596
(860) 545-0174 Fax
------------------------------
Message: 5
Date: Tue, 1 Feb 2005 15:53:32 -0000
From: "Prior, Andrew" <Andrew.Prior <@t> Smith-Nephew.com>
Subject: [Histonet] Professional Qualifications in UK
To: "'histonet <@t> lists.utsouthwestern.edu'"
<histonet <@t> lists.utsouthwestern.edu>
Message-ID: <D16CB9D969B4D3119F0D009027B70BBF09EE61A8 <@t> yofs04.eur.sn>
Content-Type: text/plain; charset="iso-8859-1"
Does anyone out there know of any professional qualifications in the UK
for
Histologists? I can find ones for Histo-paths, but I don't do any
pathology
work.
>From what I gather, there are two levels of certification in the US for
histologists and pathologists. Is the same true here in Ol' Blighty? Is
it
possible to do distance-learning for the American one?
Thanks in advance
Andrew
Andrew Prior
Histologist
Smith & Nephew Research Centre
York Science Park
Heslington
York
UK
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Message: 6
Date: Tue, 1 Feb 2005 16:11:44 -0000
From: "Prior, Andrew" <Andrew.Prior <@t> Smith-Nephew.com>
Subject: [Histonet] Re: Millers Elastin Stain HELP
To: "'histonet <@t> lists.utsouthwestern.edu'"
<histonet <@t> lists.utsouthwestern.edu>
Message-ID: <D16CB9D969B4D3119F0D009027B70BBF09EE61A9 <@t> yofs04.eur.sn>
Content-Type: text/plain; charset="iso-8859-1"
Julia ,
You can find Miller Elastin Stain on the UK Version of the Raymond Lamb
website ( http://www.ralamb.net/ ), but for some reason it's not on the
US
version of the site. You may be able to get it shipped across or just
hassle
R A Lamb in the States to sell you some.
Hope this helps
Andrew
Andrew Prior
Histologist
Smith & Nephew Research Centre
York Science Park
Heslington
York
UK
>Message: 20
>Date: Tue, 1 Feb 2005 08:13:49 -0500
>From: Celebre Julia <celebrej <@t> HHSC.CA>
>Subject: [Histonet] Millers Elastin Stain HELP
>To: "'histonet <@t> lists.utsouthwestern.edu'"
<histonet <@t> lists.utsouthwestern.edu>
>Message-ID:
<3AADFB88753AD31189C100902786B91C14E6DAE4 <@t> hch_nt_exchange.hhsc.ca>
>Content-Type: text/plain; charset="iso-8859-1"
>Good morning all....
>Our lab makes our own Millers solution, and I think it's just for plain
old
>punishment because if anyone has had to make it they understand what I
>mean... Normally the stain works beautifully, but since we've run out
of
>'Victoria Blue 4R' and have used 'Victoria Blue B' as a substitute the
stain
>doesn't seem to be working too well. The reason why we substituted
because
>Victoria Blue 4R is no longer availalbe..
>What I'm asking all you histology guru's out there...
>1. Does anybody know of a company that sells pre-made Millers?
>2. Does anybody know where we can purchase Victoria Blue 4R?
>3. Does anybody know if/why Victoria Blue B can be used as a
substitute?
>Thanks..
>Julia Celebre MLT
>Anatomic Pathology
>Hamilton General Hospital
>905-527-0271 ext46145
>email: celebrej <@t> hhsc.ca
Confidentiality.
This electronic transmission is strictly confidential to Smith & Nephew
and
intended solely for the addressee. It may contain information which is
covered by legal, professional or other privilege. If you are not the
intended addressee, or someone authorised by the intended addressee to
receive transmissions on behalf of the addressee, you must not retain,
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------------------------------
Message: 7
Date: Tue, 1 Feb 2005 11:22:18 -0500
From: "Bonner, Janet" <Janet.Bonner <@t> FLHOSP.ORG>
Subject: RE: [Histonet] yeast and bacteria on slides
To: "'Melissa Jans'" <histomjans <@t> yahoo.com>,
histonet <@t> lists.utsouthwestern.edu
Message-ID: <07AB60D5D7B9754EBF56F360F98D083DEB418B <@t> fh2k093.fhmis.net>
Content-Type: text/plain; charset="iso-8859-1"
Melissa,
We are a 24-7 operation and had the same problem when our shifts
exceeded
eight hours and some of the waterbathes were not changed out. Changing
the
waterbath out when each shift was done totally eliminated the problem.
-Janet
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu]On Behalf Of Melissa
Jans
Sent: Monday, January 31, 2005 8:40 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] yeast and bacteria on slides
I need some help!!
We have been experiencing some yeast and bacteria showing up on some of
our
special stains. It started about 2 weeks ago and seems to be sporadic
(one
day...no problems, the next day, several stains are affected). The
yeast
and bacteria (rods) are clumped together all over the slide, on the
tissue
sections, around the sections and on the slide in areas where there is
no
tissue. The stains we have seen the contamination are the Gram, PAS/D,
PAS
and GMS. All of these stains are automated except the Gram, so I do not
think the special stainer is the problem.
Here is what I have eliminated as problem areas:
-Processor solutions and paraffin (we cut and stained a block from
another
institute and had contamination)
-Waterbaths, slide drying boards, water containers (used to fill
waterbathes), stainer containers, ice pans (all of the mentioned have
been
thoroughly bleached)
-Tap water and DI water (all water supplies have been tested by sending
a
sample to micro...there was no bacteria or yeast detected)
-Stay-on used as a slide adhesive (smeared some on a slide and stained
it)
-Bad batch of slides (we have had contamination on two different slide
sources (vendors))
Does anybody have any ideas? What am I missing? Any suggestions would
be
much appreciated.
Melissa Jans
University of Iowa Hospitals and Clinics
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Message: 8
Date: Tue, 01 Feb 2005 11:51:43 -0500
From: John Kiernan <jkiernan <@t> uwo.ca>
Subject: Re: [Histonet] Formalin pigment
To: "Bruijntjes, J.P." <bruyntjes <@t> voeding.tno.nl>,
histonet <@t> lists.utsouthwestern.edu
Message-ID: <41FFB39F.954A3FB <@t> uwo.ca>
Content-Type: text/plain; charset=us-ascii
Formalin pigment is formed by the action of acid
(pH <5.6) or alkali (pH >8) on haemoglobin. According
to Lillie's big book (p. 488-489) similar pigment occurs
around sites of haemorrhage in the gastric mucosa; it's
then called HCl pigment. Malaria pigment is also
closely similar. Lillie cites his own work in the field,
which was published as 3 papers in 1947.
--
-------------------------------
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
_______________________________
"Bruijntjes, J.P." wrote:
>
> Is there anyone who can tell me what other reason could cause the
> formation of formalin pigment except a too acidic formalin?
>
>
>
> Joost Bruijntjes
>
> TNO Nutrition and Food research
>
> Zeist
>
> The Netherlands
>
>
>
> This e-mail and its contents are subject to the DISCLAIMER at
http://www.tno.nl/disclaimer/email.html
> _______________________________________________
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------------------------------
Message: 9
Date: Tue, 1 Feb 2005 23:22:22 +0600
From: Subratab <subratab <@t> bdonline.com>
Subject: [Histonet] another background / non-specific staining problem
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <200502011727.j11HRLYb031576 <@t> mailout.proshikanet.com>
Content-Type: text/plain; charset="iso-8859-1";
Dear All
I am doing immunohistochemistry for 8-OHdG (a DNA marker for
oxidative
stress) in rat kidney sections (not perfused) fixed in
methacarn.
Along with positive nuclear staining, I am getting some
background
staining in some glomerulus and also in some interstitial area.
This non-specific background is mainly in the areas where red
cells
are abundant (the tissue is not perfused). The same background is
also
present in my negative control slides (absence of primary Ab).
My protocol is
1. Deparaffinization
2. Microwave exposure for 5 min in citrate buffer (10 mM, pH 6)
3. Horse serum blocking (5%) in PBS for 1 hr at room temp
4. Primary Ab (mouse monoclonal) against 8-OHdG (N45.1) overnight at
4
C (diluent 1% BSA and 0.3% triton X 100 in PBS)
5. Horse anti-mouse secondary Ab 1 hr at room temp
6. 3% H2O2 in methanol for 10 min.
7. ABC
8. DAB
9. Counter stain with hematoxilin
10. Dehydration and mounting.
I did some experiments to identify if this non-specific staining
is
due to endogenous peroxidase/biotin.
I followed above protocol omitting both the primary and secondary
Ab.
I did not get any background stain, my slides were clean. So
I
interpreted that the background is not due to
endogenous
peroxidase/biotin. Most probably it is due to non-specific binding
of
secondary Ab with red cell (or other tissue) components.
Then I tried to block non-specific binding of secondary Ab
in
different ways (5% horse serum, 20% horse serum, 1%, and 5%
non-fat
milk, 1% BSA plus 1% non-fat milk). But I could not able to reduce
the
background. Lastly, I am writing you all for any suggestion.
Thank you in advance
Subrata Biswas
University of Campinas
SP, Brazil
------------------------------
Message: 10
Date: Tue, 1 Feb 2005 23:33:43 +0600
From: Subratab <subratab <@t> bdonline.com>
Subject: [Histonet] RE: HRP-conjugated secondary and
biotinylated-secondary
To: "C.M. van der Loos" <c.m.vanderloos <@t> amc.uva.nl>
Cc: histonet <@t> lists.utsouthwestern.edu
Message-ID: <200502011738.j11HcfYb031689 <@t> mailout.proshikanet.com>
Content-Type: text/plain; charset="iso-8859-1";
Dear van der Loos
Thank you very much for your reply with historical overview.
Thank you everybody who replyed to my question.
Subrata.
--------- Mensagem Original --------
From: "C.M. van der Loos" <c.m.vanderloos <@t> amc.uva.nl>
To: "histonet <@t> lists.utsouthwestern.edu"
<histonet <@t> lists.utsouthwestern.edu>
Cc: subratab <@t> bdonline.com
Subject: RE: HRP-conjugated secondary and biotinylated-secondary
Date: 31/01/05 14:29
Hi,
The existence of both biotinylated- and HRP-conjuated secondary
antibodies reflects the development of IHC techniques into
more staining efficiency/sensitivity. In the old days, a 2-step
staining method using a HRP-conjugated secondary anti-mouse or
anti-rabbit was common practice. Mid 80-ies the 3-step staining
method
using a biotinylated 2nd step reagent and streptavidin/HRP conjugate
or streptavidin-biotinylated HRP complex became very popular because
of its superior staining efficiency/sensitivity. The disadvantage
here
is the occurrence of endogenous biotin in many tissues. Endogenous
biotin is not present in untreated formalin-fixed and
paraffin-embedded tissue sections, but will be retrieved especially
when using Tris-EDTA pH8-9.
Only recently we have gone to a 2-step staining procedure again, now
based on both secondary antibody and HRP attached to a polymer
backbone. The efficiency/sensitivity of these polymers is comparable
with the 3-step streptavidin/HRP procedure, but avoids the problem of
endogenous biotin.
I hope this small historical overview helps.
Chris van der Loos, PhD
Dept. of Pathology
Academical Medical Center M2-230
Meibergdreef 9
NL-1105 AZ Amsterdam
The Netherlands
phone: +31 20 5665631
fax: +31 20 6960389
e-mail: [1]c.m.vanderloos <@t> amc.uva.nl
----- Original Message -----
From Subratab <subratab <@t> bdonline.com>
Date Sun, 30 Jan 2005 0:18:56 +0600
To <histonet <@t> lists.utsouthwestern.edu>
Subject [Histonet] HRP-conjugated secondary and
biotinylated-secondary
Hi,
Is it a common practice to use HRP-conjugated secondary antibody
instead of biotinylated secondary antibody for
immunohistochemistry
(with DAB developing system)? I am confused about the advantage/
disadvantage of using HRP-conjugated secondary antibody
for
immunos.
Subrata Biswas
UNICAMP, SP, Brazil.
References
1. mailto:c.m.vanderloos <@t> amc.uva.nl
------------------------------
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