[Histonet] RE: Histonet Digest, Vol 20, Issue 5
Rice, Michael
Michael.Rice <@t> holy-cross.com
Wed Jul 6 07:16:28 CDT 2005
Joe, I could not agree with you more, in these days of declining revenue and increasing costs, it seems almost criminal to discard a reagent in histology because of an arbitrary date. Having once worked for a antibody manufacturer, we determined expiration dates by subjecting the Ab to extreme high and low temperatures to accelerate the rate at which the reagent would stop working. In routine circumstances in a lab, this would not be the case. In any event, as long as the reagent passes QC each time it is used should be the determining point. Since CAP does not pay for my reagents, I feel as you do that the question should be revisited
Mike Rice
patholgy supervisor
holy cross hospital
ft lauderdale
-----Original Message-----
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Subject: Histonet Digest, Vol 20, Issue 5
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Today's Topics:
1. PCR and ISH in fixed tissues (Caroline Bass)
2. Re: PCR and ISH in fixed tissues (Agripina Suarez)
3. Re: formic acid decal (clifford berger)
4. RE: PCR and ISH in fixed tissues (Tony Henwood)
5. ER/PR staining of normal breast tissue (Patrick Paulusse)
6. non-muscle myosin IIA (Edwards, R.E.)
7. Rotating shifts... (Tom McNemar)
8. antibody search for BMP (louise renton)
9. revisiting expired immmuno reagents (Joe Nocito)
10. RE: Formic Acid decalcification (Pixley, Sarah (pixleysk))
11. Impress (Van Eyck, Deb)
12. liver cryosections (Till, Renee)
13. RE: liver cryosections (Monfils, Paul)
----------------------------------------------------------------------
Message: 1
Date: Mon, 04 Jul 2005 13:54:08 -0400
From: Caroline Bass <cbass <@t> bidmc.harvard.edu>
Subject: [Histonet] PCR and ISH in fixed tissues
To: 'histonet <@t> lists.utsouthwestern.edu'
<histonet <@t> lists.utsouthwestern.edu>
Message-ID: <9E94278E-ECB4-11D9-8BC8-0003930771EE <@t> bidmc.harvard.edu>
Content-Type: text/plain; charset=US-ASCII; format=flowed
Hey guys,
My boss is submitting a grant and he does not believe that you can do
PCR (RT) or ISH with fixed tissues. I thought you could but I don't
know any details. Could someone elaborate? Is there a particular way
the tissue should be fixed? In other words do I have to use FFPE
sections or can I use 4% paraformaldehyde perfusion?
Any advice would be helpful.
Thanks,
Caroline Bass
------------------------------
Message: 2
Date: Mon, 04 Jul 2005 11:09:57 -0700
From: "Agripina Suarez" <ASuarez <@t> mrl.ubc.ca>
Subject: Re: [Histonet] PCR and ISH in fixed tissues
To: <cbass <@t> bidmc.harvard.edu>,<Histonet <@t> lists.utsouthwestern.edu>
Message-ID: <s2c9192b.037 <@t> mail.mrl.ubc.ca>
Content-Type: text/plain; charset=US-ASCII
Hi Caroline.
Most of the ISH I do are on paraformaldehyde/formalin-fixed
paraffin-embedded sections and they work very well. The tissues are
fixed either in 4% paraformaldehyde or in commercially available 10%
neutral buffered formalin for 12 to 24 hours, depends on the size of the
tissue.
Hope this helps.
Agripina
Agripina C. Suarez
McDonald Research Laboratories
iCAPTURE Centre, St Paul's Hospital
1081 Burrard St, Vancouver
B.C., Canada V6Z 1Y6
Tel no. 604 682 2344 local 62562
Fax no. 604 806 8351
>>> Caroline Bass <cbass <@t> bidmc.harvard.edu> 7/4/2005 10:54:08 AM >>>
Hey guys,
My boss is submitting a grant and he does not believe that you can do
PCR (RT) or ISH with fixed tissues. I thought you could but I don't
know any details. Could someone elaborate? Is there a particular way
the tissue should be fixed? In other words do I have to use FFPE
sections or can I use 4% paraformaldehyde perfusion?
Any advice would be helpful.
Thanks,
Caroline Bass
_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
------------------------------
Message: 3
Date: Mon, 04 Jul 2005 16:10:06 -0400
From: clifford berger <miffordclark <@t> optonline.net>
Subject: Re: [Histonet] formic acid decal
To: patsy ruegg <pruegg <@t> ihctech.net>, lpwenk <@t> sbcglobal.net, 'louise
renton' <louise.renton <@t> gmail.com>, Histonet <@t> lists.utsouthwestern.edu
Message-ID: <000e01c580d4$5f1e71e0$0200a8c0 <@t> dellovo0ll7kuk>
Content-Type: text/plain; charset=iso-8859-1
For more information on this product, technical papers and much more information, you can visit our website, www.decal-bone.com.
You can also call us at 1-800-428-5856 for free samples.
Best regards and happy 4rth
Cliff Berger
Decal Chemical Corp
The Real Decal
1-800-428-5856
----- Original Message -----
From: "patsy ruegg" <pruegg <@t> ihctech.net>
To: <lpwenk <@t> sbcglobal.net>; "'louise renton'" <louise.renton <@t> gmail.com>; <Histonet <@t> lists.utsouthwestern.edu>
Sent: Monday, July 04, 2005 11:24 AM
Subject: RE: [Histonet] formic acid decal
> For I IHC I use ImmunoCal for Decal Chemicals, it is a 5% formic acid with
> buffers.
> Patsy
>
> -----Original Message-----
> From: histonet-bounces <@t> lists.utsouthwestern.edu
> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Lee & Peggy
> Wenk
> Sent: Friday, July 01, 2005 5:13 PM
> To: 'louise renton'; Histonet <@t> lists.utsouthwestern.edu
> Subject: RE: [Histonet] formic acid decal
>
> A milder formic acid decalcification solution is FASC = formic acid-sodium
> citrate. It's a buffered formic acid. Sections of vertebral bone take 5-7
> days to decalcify. I don't know how long a bone biopsy takes. When my
> students have to submit a bone section for their certification exam, this is
> what we use. It gives great nuclear detail, fantastic eosinophil granules.
> So I expect IHC would be equally great. We use the procedure out of the
> Bancroft book.
>
> 65 mL of 20% aqueous trisodium citrate (13 g in 65 mL water)
> 35 mL of 90% formic acid (that's full strength. Don't dilute)
> pH is about 2.3
> Change solution every 3 days.
>
> We did have a rare bone tumor, and none of the IHC worked, until we
> decalcified it in EDTA. Then it worked great. (Sorry, don't remember what
> type of tumor or what type of antibody.) But you might want to try the EDTA,
> if you have a lot of time. Vertebral bone takes 2-3 weeks. The little bone
> fragments we had from the bone tumor took 3 days. (would have been a 2-3
> hours in our usual 5% hydrochloric acid)
>
> 25 g EDTA disodium salt (ethylenediaminetetracetic acid) (note: MUST be
> disodium salt)
> 175 mL d. water
> Solution is cloudy. Add 2.5-3.0 g of sodium hydroxide until solution becomes
> clear. pH should be about 7 at that point.
> Change solution about twice a week.
>
> Peggy A. Wenk, HTL(ASCP)SLS
> William Beaumont Hospital
> Royal Oak, MI 48073
>
>
> Lee & Peggy Wenk
> -----Original Message-----
> From: histonet-bounces <@t> lists.utsouthwestern.edu
> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of louise
> renton
> Sent: Friday, July 01, 2005 8:32 AM
> To: Histonet <@t> lists.utsouthwestern.edu
> Subject: [Histonet] formic acid decal
>
> Hi all,
>
> on pain of being repetitive/lazy/slovenly/slothful/unprofessional,
> would someone PLEASE share their recipe for formic acid decalcifying
> solution suitable for samples on which IHc will be performed. Many many
> thanks
>
> --
> Louise Renton (grovelling)
> Bone Research Unit
> University of the Witwatersrand
> Johannesburg
> South Africa
> "....I know who I am. No-one else knows who I am. If I was a giraffe, and
> someone said I was a snake, I'd think no, actually I'm a giraffe"
> Richard Gere
>
> _______________________________________________
> Histonet mailing list
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> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>
> _______________________________________________
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>
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------------------------------
Message: 4
Date: Tue, 5 Jul 2005 09:10:29 +1000
From: Tony Henwood <AnthonyH <@t> chw.edu.au>
Subject: RE: [Histonet] PCR and ISH in fixed tissues
To: "'Caroline Bass'" <cbass <@t> bidmc.harvard.edu>,
"'histonet <@t> lists.utsouthwestern.edu'"
<histonet <@t> lists.utsouthwestern.edu>
Message-ID: <1CF2E2E5BB36D5119E7A0008C791F3741269E2C0 <@t> simba.kids>
Content-Type: text/plain
Caroline,
We have successfully done ISH for EBERs, Adenovirus RNA, CMV RNA,
Chromogranin mRNA, Estrogen Receptor mRNA, and nCAM RNA to name a few on
both Frozen sections and Formalin Fixed Paraffin Embedded Tissue (FFPE).
We have also successfully extracted RNA and DNA from FFPE tissues for PCR
(Ewing's and Rhabdo translocation PCR studies)and do this routinely.
Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager & Senior Scientist
The Children's Hospital at Westmead,
Locked Bag 4001, Westmead, 2145, AUSTRALIA.
Tel: 612 9845 3306
Fax: 612 9845 3318
-----Original Message-----
From: Caroline Bass [mailto:cbass <@t> bidmc.harvard.edu]
Sent: Tuesday, 5 July 2005 3:54 AM
To: 'histonet <@t> lists.utsouthwestern.edu'
Subject: [Histonet] PCR and ISH in fixed tissues
Hey guys,
My boss is submitting a grant and he does not believe that you can do
PCR (RT) or ISH with fixed tissues. I thought you could but I don't
know any details. Could someone elaborate? Is there a particular way
the tissue should be fixed? In other words do I have to use FFPE
sections or can I use 4% paraformaldehyde perfusion?
Any advice would be helpful.
Thanks,
Caroline Bass
_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
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Message: 5
Date: Sat, 4 Jun 2005 20:06:38 -0400
From: "Patrick Paulusse" <wolf <@t> webhart.net>
Subject: [Histonet] ER/PR staining of normal breast tissue
To: "Histonet" <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <000701c56962$85217100$57b05bd1 <@t> wolf>
Content-Type: text/plain; charset="iso-8859-1"
Good Evening All,
Should normal breast duct tissue always stain positively with estrogen and
progesterone antibodies. If not, is this a natural occurrence or is there
something physiologically occuring to prevent the staining. I have had
several occasions when the normal breast duct tissue will not stain (the
corresponding tumour tissue in the same block was also negative). An
external control on the same slide stains wonderfully. Fixation is never
less than 24 hours and seldom over 72 hours in 10% NBF. I have several
control blocks and use the one that mimics the fixation and processing of
the test specimen. Any comments and references are always welcome.
Thanking you in advance,
Patrick Paulusse
Anatomical Pathology
Pembroke Regional Hospital
------------------------------
Message: 6
Date: Tue, 5 Jul 2005 12:32:03 +0100
From: "Edwards, R.E." <ree3 <@t> leicester.ac.uk>
Subject: [Histonet] non-muscle myosin IIA
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<DC88BEDFD1FC3F468D0376A7C75465F705C75463 <@t> Saffron.cfs.le.ac.uk>
Content-Type: text/plain; charset="iso-8859-1"
Looking for an antibody to the above for immunohistochemistry...
Thanks
Richard Edwards
MRC TOX UNIT.....LEICESTER.....U.K........
------------------------------
Message: 7
Date: Tue, 5 Jul 2005 09:25:27 -0400
From: Tom McNemar <TMcNemar <@t> lmhealth.org>
Subject: [Histonet] Rotating shifts...
To: Histonet <histonet <@t> pathology.swmed.edu>
Message-ID:
<6CD94D97ED7D924BA5C2B588FA9528213967D9 <@t> nt_exchange.lmhealth.org>
Content-Type: text/plain; charset="iso-8859-1"
I was wondering if most histo labs use rotating shifts or if people mostly
work the same hours. Do you have people that just work one shift and
primarily do one job (gross, cut, stain, etc.)?
At full staff we have 4 techs and 3 of them rotate between 2 weeks of 6-2:30
and 1 week of 7:30-4 (surgicals). I wondered if switching the hours to a
straight 7:30 - 4 position might help attract someone. I was also thinking
that we would have a more consistency/continuity if everyone had a certain
job/area that they were primarily responsible for.
All thoughts appreciated.
Tom Mc Nemar HT(ASCP)
Histology Supervisor
Licking Memorial Hospital
Newark, Ohio 43055
------------------------------
Message: 8
Date: Tue, 5 Jul 2005 15:52:42 +0200
From: louise renton <louise.renton <@t> gmail.com>
Subject: [Histonet] antibody search for BMP
To: Histonet <@t> lists.utsouthwestern.edu
Message-ID: <e483362e050705065210e36d93 <@t> mail.gmail.com>
Content-Type: text/plain; charset=ISO-8859-1
Dear all,
I am looking for antibodies to BMP (bone morphogenic protein) 3, 6 and
7 (AKA Op1) for use on FFPE embedded tissue. I would be grateful for
any input, as an internet search has come up with very little
information. Thank you
--
Louise Renton
Bone Research Unit
University of the Witwatersrand
Johannesburg
South Africa
"....I know who I am. No-one else knows who I am. If I was a giraffe,
and someone said I was a snake, I'd think no, actually I'm a giraffe"
Richard Gere
------------------------------
Message: 9
Date: Tue, 5 Jul 2005 09:01:51 -0500
From: "Joe Nocito" <JNocito <@t> Pathreflab.com>
Subject: [Histonet] revisiting expired immmuno reagents
To: "Histonet" <histonet <@t> pathology.swmed.edu>
Message-ID: <JFEMICGBHEGPLAMIJPJPCEJFCKAA.JNocito <@t> Pathreflab.com>
Content-Type: text/plain; charset="iso-8859-1"
Hello histoland,
okay, I've been shooting my mouth off a lot lately and I want y'all to know
that I contacted the CAP about revising the question about using expired
immuno reagents.
The question in question (like that huh?) in the latest CAP checklist is
ANP.22432. Whether they contact me or not is another question (so many
questions).
My rationale for changing the question are these
1. Immuno reagents are too expensive to throw away.
2. Each time a run is performed, the reagents are revalidated
3. A laboratory must have a written procedure in place detailing the
revalidating process.
4. A quality control form must be used and signed by the medical director
assuring that the reagents work.
I am open to suggestions and other ideas that I may have over looked. With
your support, maybe we can, at least, have this question revised.
Of course, I expect some fallout because of this, but hey, it wouldn't be me
if I didn't
Enjoy!!!
Joe Nocito, BS, HT(ASCP) QIHC
Histology Manager
Pathology Reference Lab
San Antonio, TX
------------------------------
Message: 10
Date: Tue, 5 Jul 2005 10:25:23 -0400
From: "Pixley, Sarah (pixleysk)" <PIXLEYSK <@t> UCMAIL.UC.EDU>
Subject: [Histonet] RE: Formic Acid decalcification
To: "'histonet <@t> lists.utsouthwestern.edu'"
<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<E7340445E0595A418B2F8DE0AE480E200364CB1F <@t> ucmail6.ad.uc.edu>
Content-Type: text/plain
Dear Louise Renton:
Here is a method from a research lab. We use 10% formic acid, but add an ion
exchange resin that soaks up the calcium. This eliminates the need to change
solutions, but brings its own problems since the resin must be stirred. This
is faster than EDTA, but I don't know if it is faster than just a 10% formic
acid solution alone. The mixture is good for many decalcifications. Great
ICC staining.
Formic Acid Decalcification: Purchase (i.e., Fisher Scientific):
Rexyn 101 H ion exchange resin
formic acid (full strength, ~90%).
Make 6-10 liters of 10% formic acid (in double distilled water)
Add 500 g of resin
(So ~50-83 g resin per liter of 10% formic acid)
Need stir bar to keep resin suspended, so put samples in cloth bags (i.e.,
homemade cotton bags) and suspend from side. Alternatively, use lab
dessicator. Samples sit above dessicator plate while stir bar goes
underneath.
Sincerely,
Sarah Pixley, Ph.D.
Dept. of Cell Biology, Neurobiology and Anatomy
University of Cincinnati College of Medicine
Cincinnati, OH 45267-0521
------------------------------
Message: 11
Date: Tue, 5 Jul 2005 09:28:07 -0500
From: "Van Eyck, Deb" <deb.vaneyck <@t> phci.org>
Subject: [Histonet] Impress
To: "'histonet <@t> lists.utsouthwestern.edu'"
<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<E02F8B5C3450E54C87F76C7D50141C550673618E <@t> wmhmail1.phci.org>
Content-Type: text/plain; charset="iso-8859-1"
Dear-histonetters-----I have also wondered about the Impress Kit----Amos and
to otheres who tried it---was it pretty interchangeable- time wise with the
Envision , or Envision + also what about antibody dilutions? same strength?
Another immuno question for you all? is anyone doing Toxoplasmosis
staining? What antibody are you using? What protocol? Thanks much-Deb Van
Eyck
> -----Original Message-----
> From: histonet-bounces <@t> lists.utsouthwestern.edu
> [SMTP:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of
> histonet-request <@t> lists.utsouthwestern.edu
> Sent: Sunday, July 03, 2005 12:06 PM
> To: histonet <@t> lists.utsouthwestern.edu
> Subject: Histonet Digest, Vol 20, Issue 3
>
> Send Histonet mailing list submissions to
> histonet <@t> lists.utsouthwestern.edu
>
> To subscribe or unsubscribe via the World Wide Web, visit
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
> or, via email, send a message with subject or body 'help' to
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> When replying, please edit your Subject line so it is more specific
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>
>
> Today's Topics:
>
> 1. Re: Histonet Digest, Vol 19, Issue 43 (Amos Brooks)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Sat, 02 Jul 2005 16:19:45 -0400
> From: Amos Brooks <amosbrooks <@t> earthlink.net>
> Subject: [Histonet] Re: Histonet Digest, Vol 19, Issue 43
> To: histonet <@t> lists.utsouthwestern.edu
> Message-ID: <42C6F6E1.2010501 <@t> earthlink.net>
> Content-Type: text/plain; charset=ISO-8859-1; format=flowed
>
> Carla,
>
> We use both depending on the antibody. Mostly we use Envision, but
> we tried the Impress on some of the antibodies that don't label very
> intensely and did see improvement. If you compare the results of a CD2,
> CD4, CD7 or CD8 odds are you'll see some improvement. Not all the
> antibodies showed marked improvement and in some cases envision was better
> so we use Envision on most but the success we've had with Impress made it
> worth switching some of the procedures. Vector might give you a sample if
> you ask nicely. Give it a whirl and see if you like it.
>
> Best of luck,
> Amos
>
>
>
> Message: 17
> Date: Thu, 30 Jun 2005 07:34:30 -0700
> From: Carla M Conway <cmconway <@t> usgs.gov>
> Subject: [Histonet] Dako EnVision G2 or Vector ImmPRESS comments
> wanted
> To: Histonet <@t> lists.utsouthwestern.edu
> Message-ID:
> <OFF6D9EC48.FF33E03E-ON88257030.004D25F6-88257030.00501497 <@t> usgs.gov>
> Content-Type: text/plain; charset=US-ASCII
>
> Hello all,
>
> I would like any comments (pro and con) regarding Dako's EnVision G2/AP
> system or Vector's ImmPRESS kit. We are currently using EnVision + , but
> are wondering if these new systems could be even better (more sensitive).
>
> Thanks for your help,
>
> Carla Conway
>
> Western Fisheries Research Center
> 6505 NE 65th St
> Seattle, WA 98115
> ph: 206-526-6282 ext. 242
> fax: 206-526-6654
> cmconway <@t> usgs.gov
>
>
>
>
>
> ------------------------------
>
> _______________________________________________
> Histonet mailing list
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> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
> End of Histonet Digest, Vol 20, Issue 3
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Message: 12
Date: Tue, 5 Jul 2005 10:39:33 -0500
From: "Till, Renee" <TillRenee <@t> uams.edu>
Subject: [Histonet] liver cryosections
To: histonet <@t> lists.utsouthwestern.edu
Message-ID:
<EB352EB477F2064285B7B2192E37CC28091A046E <@t> exchpeds.ad.uams.edu>
Content-Type: text/plain; charset=us-ascii
Can anyone offer some advice on cutting liver cryosections? My every
attempt produces shredded tissues. I know in paraffin embedding that
means they are dry and I would soak it in ice water before I cut, but
what do you do with cryosections? I'm pretty sure it's not my cutting
technique, though I am fairly new at it. I've adjusted the roll plate
and the vacume window. And I've tried it without the roll plate, though
as we are just starting out with our cryostat I don't have any good
brushes for pulling the section. Any ideas? Maybe they did not have
enough time to come down from -80 to -20? I left them in for about 2
hours.
Renee'
------------------------------
Message: 13
Date: Tue, 5 Jul 2005 11:45:01 -0400
From: "Monfils, Paul" <PMonfils <@t> Lifespan.org>
Subject: RE: [Histonet] liver cryosections
To: "'histonet <@t> lists.utsouthwestern.edu'"
<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<09C945920A6B654199F7A58A1D7D1FDE01717565 <@t> lsexch.lsmaster.lifespan.org>
Content-Type: text/plain; charset="ISO-8859-1"
The block is too cold. -20 degrees is an appropriate temperature for
sectioning many tissues, but some tissues require a lower temperature and
others require a higher temperature. For liver, try -15 degrees. For some
samples you may have to go as high as -12 degrees.
Paul M.
> ----------
> From: histonet-bounces <@t> lists.utsouthwestern.edu on behalf of Till,
> Renee
> Sent: Tuesday, July 5, 2005 8:39 AM
> To: histonet <@t> lists.utsouthwestern.edu
> Subject: [Histonet] liver cryosections
>
> Can anyone offer some advice on cutting liver cryosections? My every
> attempt produces shredded tissues. I know in paraffin embedding that
> means they are dry and I would soak it in ice water before I cut, but
> what do you do with cryosections? I'm pretty sure it's not my cutting
> technique, though I am fairly new at it. I've adjusted the roll plate
> and the vacume window. And I've tried it without the roll plate, though
> as we are just starting out with our cryostat I don't have any good
> brushes for pulling the section. Any ideas? Maybe they did not have
> enough time to come down from -80 to -20? I left them in for about 2
> hours.
>
>
>
> Renee'
>
>
>
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>
------------------------------
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