[Histonet] RE: Fluorescent Illumination Systems: Suggestions please?
Glenn Smith
gsmith <@t> confocal.com
Tue Sep 27 09:06:48 CDT 2005
Melissa,
If you haven't already, you may also want to post your question to a
microscopy listserve too. For example, try
http://listserv.acsu.buffalo.edu/cgi-bin/wa?A0=confocal.
Glenn Smith, P.Eng.
519.886.9013 x38 (office)
519.498.0614 (mobile)
Biomedical Photometrics Inc/GeneFocus <www.confocal.com OR
www.GeneFocus.com>
Fluorescence and Brightfield Laser Scanning Instruments and Software for
Pathology
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of
histonet-request <@t> lists.utsouthwestern.edu
Sent: Tuesday, September 27, 2005 12:48 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: Histonet Digest, Vol 22, Issue 33
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Today's Topics:
1. RE: antigen retrieval for IHC (May Wei)
2. RE: antigen retrieval for IHC (Morken, Tim - Labvision)
3. Fluorescent Illumination Systems: Suggestions please?
(Melissa Gonzalez)
4. Re: cryostat decontamination (Instrumedics)
5. RE: antigen retrieval for IHC (Pamela Marcum)
6. Low temp Antigen Retrieval for Bone Sections (Kim Merriam)
7. Re: Fluorescent Illumination Systems: Suggestions please?
(kgibbon <@t> qltinc.com)
8. RE: Low temp Antigen Retrieval for Bone Sections (Monfils, Paul)
9. cpt codes for direct smears (Barb Richmond)
10. Re: antigen retrieval for IHC (Bryan Hewlett)
11. slides and RE: [Histonet] Low temp Antigen Retrieval for Bone
Sections (Gayle Callis)
12. RE: slide QC (Baez, Janet)
13. Cryostat decontamination (Jan.Minshew <@t> leica-microsystems.com)
14. RE: Posting for Histology List Serve (Nelson, Sandy - PPH)
15. Staining fibrin - thrombus (Andrea T. Hooper)
16. IHC background problems on chicken embryos (Katri Tuomala)
17. Pink precipitate is monoformazan from BCIP/NBT reaction on
ISH? (Ralph Puchalski)
18. Re: Microwave PROCCESSING (Histology)
19. Re: Embedding centers (Histology)
20. section edges lifting (Sam Vaughn)
21. Re: Pink precipitate is monoformazan from BCIP/NBT reaction
onISH? (John Kiernan)
----------------------------------------------------------------------
Message: 1
Date: Mon, 26 Sep 2005 10:05:15 -0700
From: "May Wei" <m.wei <@t> biogenex.com>
Subject: RE: [Histonet] antigen retrieval for IHC
To: <jkiernan <@t> uwo.ca>
Cc: histonet <@t> lists.utsouthwestern.edu
Message-ID:
<37DC9F93CF7F864182D0463EF93D571B0B5043 <@t> ISLETON2.california.biogenex.com>
Content-Type: text/plain; charset="us-ascii"
One of papers with Shi/Taylor is "Standardization of Immunohistochemistry
Based on Antigen Retrieval Technique for Routine Formalin-fixed Tissue
Sections" in Applied Immunohistochemistry 6(2): 89-96, 1998. I have the
article. I also have the list of AR paper references. If you like to receive
it, I can send to you.
May Wei
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of John Kiernan
Sent: Sunday, September 25, 2005 11:14 PM
To: Maria Mejia
Cc: histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] antigen retrieval for IHC
Maria, can you give chapter and verse for the Shi/Taylor and Puchrler/Meloan
papers? Most papers with Shi and Taylor among the authors are about high
temperature antigen retrieval (boiling water, with pH optima for various
antigens).
Holde Puchtler and Susan Meloan published many imortant papers about
staining ad histochemistry in the 1970s=1980s. Susan M was a histonetter in
the 1990s.
*****************
Maria Mejia wrote:
>
> Hello,
>
> I believe it was sometime in June of this year that there was a
> discussion on the histonet regarding "the simplest form" of reversing
> the effects for formalin fixation on tissue was to wash the tissue
> well (before) processing.
>
> Well, I just read the article "Antigen retrieval IHC: Past, Present, &
> Future by Shan-Rong Shi, richard J. Cote & Clive R. Taylor (can google
> this article). It's a very interesting! Anyway, the article has a
> section titled non-heating AR method stating (this) same simple method
> by (Puchtler & Meloan). It also goes on to say that Elias JM (1990)
> adopted this method routinely by storing deparaffinized in fresh
> changes of 10% sucrose/PBS @ 4C overnight (before) IHC.
>
> My questions are, does anyone know or tried this method used by Elias?
> And, can anyone explain the mechanism of 10% sucrose/PBS play in this
> AR method?
>
> Just curious!
>
> Maria Bartola Mejia
> Smith-Kettlewell Eye Research Institute San Francisco, CA 94115
> Email: maria <@t> ski.org
> Phone: (415)-345-2185
>
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
------------------------------
Message: 2
Date: Mon, 26 Sep 2005 13:12:32 -0400
From: "Morken, Tim - Labvision" <tpmorken <@t> labvision.com>
Subject: RE: [Histonet] antigen retrieval for IHC
To: histonet <@t> lists.utsouthwestern.edu
Message-ID:
<0556BE8AC5551E4E8AF6BB9E42509BA20328D178 <@t> usca0082k08.labvision.apogent.com>
Content-Type: text/plain
It's true that most papers by Shi et al are on high-temperature antigen
retrieval, but in their book on antigen retrieval (Antigen retrieval
Techniques, Immunohistochemistry and Molecular Morphology, ed. Shan-Rong Shi
et al, Eaton Publishing, 2000) they make clear that the term "antigen
retrieval" should cover all types -enzyme, heat, or other. In fact they make
a plea for everyone publishing methods to use the term "antigen retrieval"
so matter what the method so that databases are easy to search. With the
plethora of terms used to decribe the methods it is nearly impossible to
find all the methods developed in the past 14 years. Part of the reason for
people using all the different terms was the assumption by many that
BioGenex had trademarked the term. BioGenex did apply for a trademark, but
then withdrew it. So the term is open to all to use. However, BioGenex does
have a patent on a very specific method of antigen retrieval, and some
antigen retrieval buffer formulations.
Tim Morken
Lab Vision - Neomarkers
www.labvision.com
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of John Kiernan
Sent: Sunday, September 25, 2005 11:14 PM
To: Maria Mejia
Cc: histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] antigen retrieval for IHC
Maria, can you give chapter and verse for the Shi/Taylor
and Puchrler/Meloan papers? Most papers with Shi and Taylor among the
authors are about high temperature antigen retrieval (boiling water, with pH
optima for various antigens).
Holde Puchtler and Susan Meloan published many imortant papers about
staining ad histochemistry in the 1970s=1980s. Susan M
was a histonetter in the 1990s.
*****************
Maria Mejia wrote:
>
> Hello,
>
> I believe it was sometime in June of this year that there was a
> discussion on the histonet regarding "the simplest form" of reversing
> the effects for formalin fixation on tissue was to wash the tissue
> well (before) processing.
>
> Well, I just read the article "Antigen retrieval IHC: Past, Present, &
> Future by Shan-Rong Shi, richard J. Cote & Clive R. Taylor (can google
> this article). It's
> a very interesting! Anyway, the article has a section titled non-heating
> AR method stating (this) same simple method by (Puchtler & Meloan). It
also
> goes on to say that Elias JM (1990) adopted this method routinely by
storing
> deparaffinized in fresh changes of 10% sucrose/PBS @ 4C overnight
> (before) IHC.
>
> My questions are, does anyone know or tried this method used by Elias?
> And, can anyone explain the mechanism of 10% sucrose/PBS play in this
> AR method?
>
> Just curious!
>
> Maria Bartola Mejia
> Smith-Kettlewell Eye Research Institute
> San Francisco, CA 94115
> Email: maria <@t> ski.org
> Phone: (415)-345-2185
>
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
------------------------------
Message: 3
Date: Mon, 26 Sep 2005 10:50:34 -0700
From: "Melissa Gonzalez" <Melissa.Gonzalez <@t> cellgenesys.com>
Subject: [Histonet] Fluorescent Illumination Systems: Suggestions
please?
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<A6B0506C2B000E45A7556B9A93C4554715B31D <@t> hqsvr01mail.cgi.com>
Content-Type: text/plain; charset="iso-8859-1"
Hello all,
Sorry to re-post, but I only received one response to the following: I am
currently in the market to upgrade our existing HB0 100 lamp house, which is
very old, to several of new systems. Either the newest version of the HB0
100 from Zeiss, a mercury halide system called X-Cite from EXFO, or a xenon
based burner called Lambda LS from Sutter Instruments. They are all
comparable in price, but my main concern is increasing the brightness of my
signal and evenly illuminating the field of view. I mostly look at DAPI,
eGFP, FITC, and Alexa 594, which from spectral outputs, each one of these
systems appears to have a different weakness in one of the above. On a plus,
the Xcite rates their bulbs at 1500 hours, and the Lambda at 500-1000hrs.
Does anyone out there have any input/comments/experience with these systems?
(This would be hooked up to a Zeiss Axioplan) Please let me know if you have
any info, as I have been unsuccessful so far with demos and need to place an
order soon.
Thanks a lot -I really appreciate it,
Melissa
------------------------------
Message: 4
Date: Mon, 26 Sep 2005 13:53:16 -0400
From: "Instrumedics" <info <@t> instrumedics.com>
Subject: Re: [Histonet] cryostat decontamination
To: <Traczyk7 <@t> aol.com>
Cc: HistoNet Server <HistoNet <@t> Pathology.swmed.edu>
Message-ID: <01d401c5c2c3$336ba0f0$6401a8c0 <@t> INSTRUMEDICS22>
Content-Type: text/plain; format=flowed; charset="iso-8859-1";
reply-type=original
Instrumedics' Cryo-Vac-Away keeps the cryostat free of trimming debris. The
debris is suctioned away as it is generated at the block face when you trim
the block.
The debris is captured in a primary filter that is inside the cryostat and
downstream of the primary filter is a viral/ bacterial filter that
captures pathogens.
The cryostat remains spotless and substantially reduces the hazards
associated with frozen sectioning.
Please visit www.instrumedics.com for details.
Bernice
----- Original Message -----
From: <Traczyk7 <@t> aol.com>
To: <histonet <@t> lists.utsouthwestern.edu>
Sent: Monday, September 26, 2005 10:59 AM
Subject: Re: [Histonet] cryostat decontamination
> Greetings,
> I recently attended Gloria Limetti's seminar at NSH about cryostat and
> microtome features. She is from the University of Pitssburgh. Attendees
> were
> given an extensive spreadsheet listing which features were available
> on
> which
> models. In an effort not to come across as non vendor specific, Gloria
> assigned all the companies letters. Just looking over the features, it's
> fairly
> easy to decipher which company is which. I don't have my copy in front
> of me
> now but I believe there are 7 models with various types of
> decontamination
> systems offered.
> Hacker Instruments SL5000 is one of the units that is available with an
> automatic decontamination feature, perhaps Gloria will post the names of
> the
> other companies.
> As for UV decontamination, it is my understanding the the UV light is only
> effective on the surfaces of the cryostat and microtome chamber where the
> light
> actually comes into contact. Nooks and cranies would still need to be
> wiped down manually. As with Vinnie, I would be interested in reading a
> study or
> two on it's effectiveness in histology applications.
> Regards,
>
> Dorothy Murphy Traczyk
> National Sales Manager
> Hacker Instruments & Industries Inc.
> PO Box 1176
> Winnsboro, SC 29180
> 1-800-442-2537
> hackerlab <@t> aol.com
> _www.hacker_ (http://www.hacker/) insruments.com
>
>
------------------------------
Message: 5
Date: Mon, 26 Sep 2005 14:01:50 -0400
From: Pamela Marcum <pmarcum <@t> vet.upenn.edu>
Subject: RE: [Histonet] antigen retrieval for IHC
To: "Morken, Tim - Labvision" <tpmorken <@t> labvision.com>,
histonet <@t> lists.utsouthwestern.edu
Message-ID: <6.1.1.1.2.20050926140018.01a53ce0 <@t> mail.vet.upenn.edu>
Content-Type: text/plain; charset="us-ascii"; format=flowed
Thanks for the update Tim. I know for years we have all been careful about
using antigen retrieval due to the BioGenex issue. They do have a patent
on the one tyoe and it was spread to all for a while.
Pam Marcum
At 01:12 PM 9/26/2005, Morken, Tim - Labvision wrote:
>It's true that most papers by Shi et al are on high-temperature antigen
>retrieval, but in their book on antigen retrieval (Antigen retrieval
>Techniques, Immunohistochemistry and Molecular Morphology, ed.
>Shan-Rong Shi et al, Eaton Publishing, 2000) they make clear that the
>term "antigen retrieval" should cover all types -enzyme, heat, or
>other. In fact they make a plea for everyone publishing methods to use
>the term "antigen retrieval" so matter what the method so that
>databases are easy to search. With the plethora of terms used to
>decribe the methods it is nearly impossible to find all the methods
>developed in the past 14 years. Part of the reason for people using all
>the different terms was the assumption by many that BioGenex had
>trademarked the term. BioGenex did apply for a trademark, but then
>withdrew it. So the term is open to all to use. However, BioGenex does
>have a patent on a very specific method of antigen retrieval, and some
>antigen retrieval buffer formulations.
>
>Tim Morken
>Lab Vision - Neomarkers
>www.labvision.com
>
>
>-----Original Message-----
>From: histonet-bounces <@t> lists.utsouthwestern.edu
>[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of John
>Kiernan
>Sent: Sunday, September 25, 2005 11:14 PM
>To: Maria Mejia
>Cc: histonet <@t> lists.utsouthwestern.edu
>Subject: Re: [Histonet] antigen retrieval for IHC
>
>
>Maria, can you give chapter and verse for the Shi/Taylor
>and Puchrler/Meloan papers? Most papers with Shi and Taylor among the
>authors are about high temperature antigen retrieval (boiling water,
>with pH optima for various antigens).
>
>Holde Puchtler and Susan Meloan published many imortant papers about
>staining ad histochemistry in the 1970s=1980s. Susan M was a
>histonetter in the 1990s.
>
>*****************
>
>Maria Mejia wrote:
> >
> > Hello,
> >
> > I believe it was sometime in June of this year that there was a
> > discussion on the histonet regarding "the simplest form" of
> > reversing the effects for formalin fixation on tissue was to wash
> > the tissue well (before) processing.
> >
> > Well, I just read the article "Antigen retrieval IHC: Past, Present,
> > & Future by Shan-Rong Shi, richard J. Cote & Clive R. Taylor (can
> > google this article). It's a very interesting! Anyway, the article
> > has a section titled non-heating AR method stating (this) same
> > simple method by (Puchtler & Meloan). It
>also
> > goes on to say that Elias JM (1990) adopted this method routinely by
>storing
> > deparaffinized in fresh changes of 10% sucrose/PBS @ 4C overnight
> > (before) IHC.
> >
> > My questions are, does anyone know or tried this method used by
> > Elias? And, can anyone explain the mechanism of 10% sucrose/PBS play
> > in this AR method?
> >
> > Just curious!
> >
> > Maria Bartola Mejia
> > Smith-Kettlewell Eye Research Institute
> > San Francisco, CA 94115
> > Email: maria <@t> ski.org
> > Phone: (415)-345-2185
> >
> > _______________________________________________
> > Histonet mailing list
> > Histonet <@t> lists.utsouthwestern.edu
> > http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>_______________________________________________
>Histonet mailing list
>Histonet <@t> lists.utsouthwestern.edu
>http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>_______________________________________________
>Histonet mailing list
>Histonet <@t> lists.utsouthwestern.edu
>http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Best Regards,
Pamela A Marcum
Manager, Histology Special Procedures
University of Pennsylvania
School of Veterinary Medicine
R.S. Reynolds Jr. CORL
New Bolton Center
382 West Street Road
Kennett Square, PA 19348
Phone - 610-925-6278
Fax - 610-925-8120
E-mail - pmarcum <@t> vet.upenn.edu
------------------------------
Message: 6
Date: Mon, 26 Sep 2005 11:34:06 -0700 (PDT)
From: Kim Merriam <kmerriam2003 <@t> yahoo.com>
Subject: [Histonet] Low temp Antigen Retrieval for Bone Sections
To: Histonet <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <20050926183406.77615.qmail <@t> web50312.mail.yahoo.com>
Content-Type: text/plain; charset=iso-8859-1
Hello All,
I will be performing some IHC on mouse femurs (about 12 or so different
antibodies). In the past, I have had a lot of trouble keeping these
sections on the slides during HIER. I did a quick search on the archives
and there were lots of suggestions, but nothing definitive.
I was reading an article about "low temp AR", overnight at 60C in TEG
buffer, pH 9.0. Has anyone tried this method? Also - what does TEG buffer
stand for?
Any help would be greatly appreciated.
Kim
Kim Merriam
Novartis
Cambridge, MA __________________________________________________
Do You Yahoo!?
Tired of spam? Yahoo! Mail has the best spam protection around
http://mail.yahoo.com
------------------------------
Message: 7
Date: Mon, 26 Sep 2005 12:22:30 -0700
From: kgibbon <@t> qltinc.com
Subject: Re: [Histonet] Fluorescent Illumination Systems: Suggestions
please?
To: Histonet <@t> lists.utsouthwestern.edu
Message-ID:
<OF6F010936.597BB3B1-ON88257088.0069C370-88257088.006A6E1F <@t> qltinc.com>
Content-Type: text/plain; charset="US-ASCII"
Hi Melissa,
I have used the Sutter xenon Lambda unit for a few years now and really like
it, The spectrum is much more even than a standard mercury bulb. I have the
model with the extended bandwidth so I could use it well into the UV
wavelengths but it does produce ozone which must be vented, I believe they
have another model that does not do this but has a higher minimum
wavelength. Your choice, depending what fluorochromes you want to light up.
Kevin Gibbon
QLT Inc.
|---------+------------------------------------------->
| | "Melissa Gonzalez" |
| | <Melissa.Gonzalez <@t> cellgenesys.co|
| | m> |
| | Sent by: |
| | <histonet-bounces <@t> lists.utsouthw|
| | estern.edu> |
| | |
| | |
| | 09/26/2005 10:50 AM |
| | |
|---------+------------------------------------------->
>---------------------------------------------------------------------------
---------------------------------------------------|
|
|
| To: <histonet <@t> lists.utsouthwestern.edu>
|
| cc:
|
| Subject: [Histonet] Fluorescent Illumination Systems: Suggestions
please? |
>---------------------------------------------------------------------------
---------------------------------------------------|
Hello all,
Sorry to re-post, but I only received one response to the following: I am
currently in the market to upgrade our existing HB0 100 lamp house, which is
very old, to several of new systems. Either the newest version of the HB0
100 from Zeiss, a mercury halide system called X-Cite from EXFO, or a xenon
based burner called Lambda LS from Sutter Instruments. They are all
comparable in price, but my main concern is increasing the brightness of my
signal and evenly illuminating the field of view. I mostly look at DAPI,
eGFP, FITC, and Alexa 594, which from spectral outputs, each one of these
systems appears to have a different weakness in one of the above. On a plus,
the Xcite rates their bulbs at 1500 hours, and the Lambda at 500-1000hrs.
Does anyone out there have any input/comments/experience with these systems?
(This would be hooked up to a Zeiss Axioplan) Please let me know if you have
any info, as I have been unsuccessful so far with demos and need to place an
order soon.
Thanks a lot -I really appreciate it,
Melissa
_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
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------------------------------
Message: 8
Date: Mon, 26 Sep 2005 15:59:28 -0400
From: "Monfils, Paul" <PMonfils <@t> Lifespan.org>
Subject: RE: [Histonet] Low temp Antigen Retrieval for Bone Sections
To: "'histonet <@t> lists.utsouthwestern.edu'"
<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<09C945920A6B654199F7A58A1D7D1FDE017175E3 <@t> lsexch.lsmaster.lifespan.org>
Content-Type: text/plain; charset="iso-8859-1"
I haven't tried this method of antigen retrieval, but TEG buffer is
Tris-EGTA buffer. The usual formula (I have seen a couple of variations) is:
Distilled water 1,000 ml
Trisma base 1.21 gram
EGTA 0.19 gram
The usual target pH is between 8.95 and 9.0
EGTA is ethylene glycol bis(B-aminoethyl)-N,N,N',N' tetraacetic acid.
(The capital "B" in the above name represents "beta". My email program
doesn't support the symbol.)
> ----------
> From: histonet-bounces <@t> lists.utsouthwestern.edu on behalf of Kim
> Merriam
> Sent: Monday, September 26, 2005 11:34 AM
> To: Histonet
> Subject: [Histonet] Low temp Antigen Retrieval for Bone Sections
>
> Hello All,
>
> I will be performing some IHC on mouse femurs (about 12 or so
> different antibodies). In the past, I have had a lot of trouble
> keeping these sections on the slides during HIER. I did a quick
> search on the archives and there were lots of suggestions, but nothing
> definitive.
>
> I was reading an article about "low temp AR", overnight at 60C in TEG
> buffer, pH 9.0. Has anyone tried this method? Also - what does TEG
> buffer stand for?
>
> Any help would be greatly appreciated.
>
> Kim
>
>
> Kim Merriam
> Novartis
> Cambridge, MA __________________________________________________
> Do You Yahoo!?
> Tired of spam? Yahoo! Mail has the best spam protection around
> http://mail.yahoo.com
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>
------------------------------
Message: 9
Date: Mon, 26 Sep 2005 15:29:00 -0500
From: "Barb Richmond" <Barb.Richmond <@t> mckennan.org>
Subject: [Histonet] cpt codes for direct smears
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<B3C5DE64612311468D8527D64FB760A4C26B70 <@t> MCK116.averamail.net>
Content-Type: text/plain; charset="iso-8859-1"
I need to know what cpt code to use for a direct smear that is made from
tissue and stained on the quick frozen section stain line. Any suggestions??
------------------------------
Message: 10
Date: Mon, 26 Sep 2005 16:31:28 -0400
From: "Bryan Hewlett" <bhewlett <@t> cogeco.ca>
Subject: Re: [Histonet] antigen retrieval for IHC
To: "Patsy Ruegg" <pruegg <@t> ihctech.net>, <jkiernan <@t> uwo.ca>, "'Maria
Mejia'" <maria <@t> ski.org>
Cc: histonet <@t> lists.utsouthwestern.edu
Message-ID: <002101c5c2d9$467fce80$6400a8c0 <@t> mainbox>
Content-Type: text/plain; format=flowed; charset="iso-8859-1";
reply-type=original
Patsy,
You will need to wash MUCH longer than 2 hours in running tap water in order
to obtain significant reversal after I year in NBF.
The Helander paper quoted in my NSH handout gives about 6 days to effect a
90% reversal and that was following 24 hour fixation.
Best regards,
Bryan
----- Original Message -----
From: "Patsy Ruegg" <pruegg <@t> ihctech.net>
To: <jkiernan <@t> uwo.ca>; "'Maria Mejia'" <maria <@t> ski.org>
Cc: <histonet <@t> lists.utsouthwestern.edu>
Sent: Monday, September 26, 2005 11:58 AM
Subject: RE: [Histonet] antigen retrieval for IHC
> Shi mentions in his book on pg.10 ANTIGEN RESTORATION
> "Simply bathing deparaffinized sections in a cold 20% sucrose-saline
> solution could, over several days, restore a certain amount of
> immunoreactivity."
>
> In Introduction to IHC by Polak and Van Noorden on pg 24 3.8.1 "The
> simplest
> form of reversing the effects of formalin is to wash the tissue well
> before
> processing"
>
> I am about to test these statements, as I just received tissues for an
> IHC project that have been in 10% NBF for over one year. I washed the
> tissues in running tap h20 for 2 hrs., I will now process them into
> paraffin. I am planning as well to put some of the sections in 20%
> sucrose at 4dc for 2 days if I have trouble getting good IHC signal.
> I will keep you all posted
> on this experiment. These are samples of mouse mammory tissue. I will be
> testing them with cleaved caspase 3 and Ki67 to start. I have both of
> these
> antibodies worked out very well for ffpe tissues fixed 24-48 hrs.
>
> Patsy
>
>
> Patsy Ruegg, HT(ASCP)QIHC
> IHCtech, LLC
> Fitzsimmons BioScience Park
> 12635 Montview Blvd. Suite 216
> Aurora, CO 80010
> P-720-859-4060
> F-720-859-4110
> wk email pruegg <@t> ihctech.net
> web site www.ihctech.net
>
>
> This email is confidential and intended solely for the use of the
> Person(s)
> ('the intended recipient') to whom it was addressed. Any views or opinions
> presented are solely those of the author. It may contain information that
> is
> privileged & confidential within the meaning of applicable law.
> Accordingly
> any dissemination, distribution, copying, or other use of this message, or
> any of its contents, by any person other than the intended recipient may
> constitute a breach of civil or criminal law and is strictly prohibited.
> If
> you are NOT the intended recipient please contact the sender and dispose
> of
> this e-mail as soon as possible.
>
>
> -----Original Message-----
> From: John Kiernan [mailto:jkiernan <@t> uwo.ca]
> Sent: Sunday, September 25, 2005 11:14 PM
> To: Maria Mejia
> Cc: pruegg <@t> ihctech.net; histonet <@t> lists.utsouthwestern.edu
> Subject: Re: [Histonet] antigen retrieval for IHC
>
> Maria, can you give chapter and verse for the Shi/Taylor and
> Puchrler/Meloan
> papers? Most papers with Shi and Taylor among the authors are about high
> temperature antigen retrieval (boiling water, with pH optima for various
> antigens).
>
> Holde Puchtler and Susan Meloan published many imortant papers about
> staining ad histochemistry in the 1970s=1980s. Susan M was a
> histonetter in the 1990s.
>
> *****************
>
> Maria Mejia wrote:
>>
>> Hello,
>>
>> I believe it was sometime in June of this year that there was a
>> discussion on the histonet regarding "the simplest form" of reversing
>> the effects for formalin fixation on tissue was to wash the tissue
>> well (before) processing.
>>
>> Well, I just read the article "Antigen retrieval IHC: Past, Present,
>> & Future by Shan-Rong Shi, richard J. Cote & Clive R. Taylor (can
>> google this article). It's a very interesting! Anyway, the article
>> has a section titled non-heating AR method stating (this) same simple
>> method by (Puchtler & Meloan). It also goes on to say that Elias JM
>> (1990) adopted this method routinely by storing deparaffinized in
>> fresh changes of 10% sucrose/PBS @ 4C overnight (before) IHC.
>>
>> My questions are, does anyone know or tried this method used by
>> Elias? And, can anyone explain the mechanism of 10% sucrose/PBS play
>> in this AR method?
>>
>> Just curious!
>>
>> Maria Bartola Mejia
>> Smith-Kettlewell Eye Research Institute San Francisco, CA 94115
>> Email: maria <@t> ski.org
>> Phone: (415)-345-2185
>>
>> _______________________________________________
>> Histonet mailing list
>> Histonet <@t> lists.utsouthwestern.edu
>> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
------------------------------
Message: 11
Date: Mon, 26 Sep 2005 15:04:48 -0600
From: Gayle Callis <gcallis <@t> montana.edu>
Subject: slides and RE: [Histonet] Low temp Antigen Retrieval for Bone
Sections
To: "Monfils, Paul" <PMonfils <@t> Lifespan.org>,
Histonet <@t> lists.utsouthwestern.edu
Message-ID:
<6.0.0.22.1.20050926150104.01b40890 <@t> gemini.msu.montana.edu>
Content-Type: text/plain; charset="us-ascii"; format=flowed
I strongly suggest if you try this retrieval, that you use Erie Scientific
EXCEL slides, which are coated specially to reduce the effects of chelator
based retrieval solutions at high aka alkaline pH. Many people experience
section loss with this pH and chelator, so Erie developed a new positively
charged coating to counteract losing precious sections. You can access
samples from them to see if they work out for you, contact your Erie sales
rep or even Erie directly, they have a website.
Biogenex also has a decalcified bone retrieval solution, another one to try
out.
At 01:59 PM 9/26/2005, you wrote:
>I haven't tried this method of antigen retrieval, but TEG buffer is
>Tris-EGTA buffer. The usual formula (I have seen a couple of
>variations) is:
>
>Distilled water 1,000 ml
>Trisma base 1.21 gram
>EGTA 0.19 gram
>
>The usual target pH is between 8.95 and 9.0
>
>EGTA is ethylene glycol bis(B-aminoethyl)-N,N,N',N' tetraacetic acid.
>
>(The capital "B" in the above name represents "beta". My email program
>doesn't support the symbol.)
>
> > ----------
> > From: histonet-bounces <@t> lists.utsouthwestern.edu on behalf of Kim
> > Merriam
> > Sent: Monday, September 26, 2005 11:34 AM
> > To: Histonet
> > Subject: [Histonet] Low temp Antigen Retrieval for Bone Sections
> >
> > Hello All,
> >
> > I will be performing some IHC on mouse femurs (about 12 or so
> > different antibodies). In the past, I have had a lot of trouble
> > keeping these sections on the slides during HIER. I did a quick
> > search on the archives and there were lots of suggestions, but
> > nothing definitive.
> >
> > I was reading an article about "low temp AR", overnight at 60C in
> > TEG buffer, pH 9.0. Has anyone tried this method? Also - what does
> > TEG buffer stand for?
> >
> > Any help would be greatly appreciated.
> >
> > Kim
> >
> >
> > Kim Merriam
> > Novartis
> > Cambridge, MA __________________________________________________
> > Do You Yahoo!?
> > Tired of spam? Yahoo! Mail has the best spam protection around
> > http://mail.yahoo.com
> > _______________________________________________
> > Histonet mailing list
> > Histonet <@t> lists.utsouthwestern.edu
> > http://lists.utsouthwestern.edu/mailman/listinfo/histonet
> >
> >
>
>_______________________________________________
>Histonet mailing list
>Histonet <@t> lists.utsouthwestern.edu
>http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)
------------------------------
Message: 12
Date: Mon, 26 Sep 2005 14:08:53 -0700
From: "Baez, Janet" <jbaez <@t> interscopepath.com>
Subject: RE: [Histonet] slide QC
To: "Bernadette Weston" <bernaweston <@t> hotmail.com>,
<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<9E956D8FEB06C2408B08AC16498325E90139FA <@t> adsl-67-113-77-28.dsl.lsan03.pacbell
.net>
Content-Type: text/plain; charset="us-ascii"
We run one slide of an appendix everyday and date the slide. The
histologist evaluates the slide before running the rest of the workload.
The Pathologists then documents the quality on our QC sheet.
-----Original Message-----
From: Bernadette Weston [mailto:bernaweston <@t> hotmail.com]
Sent: Monday, September 26, 2005 4:21 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] slide QC
What method of QC checking slides(H&E) do you use, does the histologist
check the slides microscopically before they take them to the
pathologist or
does the pathologist review them and give either a verbal or written
evaluation of the day's work? If the histologist does the before check,
how
many slides are reviewed?
Bernadette Weston HT
Histology Supervisor
Barberton Citizens Hospital
Barberton,OH
_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
------------------------------
Message: 13
Date: Mon, 26 Sep 2005 17:14:38 -0500
From: Jan.Minshew <@t> leica-microsystems.com
Subject: [Histonet] Cryostat decontamination
To: Traczyk7 <@t> aol.com
Cc: histonet <@t> lists.utsouthwestern.edu,
histonet-bounces <@t> lists.utsouthwestern.edu
Message-ID:
<OFBC52FF04.6AFDB74B-ON86257088.0056B4DC-86257088.007A30AA <@t> leica-microsystem
s.com>
Content-Type: text/plain; charset=ISO-8859-1
Hi Dorothy and Histonet subscribers,
I'm writing in response to comments I've seen in the past few days about UV
disinfection procedures for cryostats. I'm not sure if the listing on the
spreadsheet from Gloria's workshop referred to our instrument or not, but
Leica recently released the CM1850 UV cryostat with ultraviolet light (UVC)
disinfection...so I thought I should chime in.
We had the same questions about cryostat disinfection that many of you have
had. Therefore, as part of our development process we hired an independent
laboratory to perform tests that would verify the efficacy of UVC exposure
against specific pathogens in the CM1850 at cold temperatures. The results
assured us that our system is a convenient and safe means of inactivating
microorganisms in the air and on exposed surfaces at temperatures down to
-20°C and that using the system significantly reduces the risk of infection
to the operator. We proudly provide a CD that contains the certificate
from the consultant, details of how the tests were performed and the
results that were obtained.
Whether a cryostat has a built-in disinfection system of any kind or not,
there are several very important things to remember about disinfecting
cryostats.
1. Before beginning a disinfection protocol, don personal protective
equipment (puncture and penetration resistant gloves, gowns, etc).
2. Remove all debris from the cryostat and disposed of it according
to the policies and procedures of your institution. The debris must be
removed because organic material (blood and proteins) may contain high
concentrations of microorganisms and could possibly inactivate chemical
germicides or prevent access to contaminated surfaces.
3. Use 70% ethyl alcohol to clean the cryostat. The germicidal
activity of ethyl alcohol is most effective in that range and it has an
advantage over isopropyl alcohol of being able to kill hydrophilic viruses.
4. For those of us in the USA (other countries have access to other
products), the EPA maintains a list of Antimicrobial Chemical/Registration
Number Indexes and it is posted on their website
http://www.epa.gov/oppad001/chemregindex.htm. From this link you can find
agents effective against bloodborne pathogens such as Mycobacterium
tuberculosis, human HIV-1 virus, Hepatitis B or Hepatitis C virus. It is
critical to remember that NONE of these solutions have been tested at low
temperatures and they can only be used at room temperature.
5. Do not create aerosols by spraying disinfectant (or anything
else) in an open cryostat chamber. Pour disinfectants onto absorbent
disposable towels and allow them to remain in contact with contaminated
surfaces for the length of time specified in the instructions of the
individual agents.
I hope this information is useful. Please let me know if you have any
questions.
Best wishes to all,
Jan Minshew HT, HTL(ASCP)
Marketing Manager
Leica Microsystems, Inc.
2345 Waukegan Rd
Bannockburn, IL 60015
800.248.0123 x7051
Traczyk7 <@t> aol.com
Sent by: To:
histonet <@t> lists.utsouthwestern.edu
histonet-bounces <@t> lists.utsouth cc: (bcc:
Jan Minshew/USDER/West/Leica)
western.edu Subject: Re:
[Histonet] cryostat decontamination
09/26/2005 09:59 AM
Greetings,
I recently attended Gloria Limetti's seminar at NSH about cryostat and
microtome features. She is from the University of Pitssburgh. Attendees
were
given an extensive spreadsheet listing which features were available on
which
models. In an effort not to come across as non vendor specific, Gloria
assigned all the companies letters. Just looking over the features, it's
fairly
easy to decipher which company is which. I don't have my copy in front of
me
now but I believe there are 7 models with various types of decontamination
systems offered.
Hacker Instruments SL5000 is one of the units that is available with an
automatic decontamination feature, perhaps Gloria will post the names of
the
other companies.
As for UV decontamination, it is my understanding the the UV light is only
effective on the surfaces of the cryostat and microtome chamber where the
light
actually comes into contact. Nooks and cranies would still need to be
wiped down manually. As with Vinnie, I would be interested in reading a
study or
two on it's effectiveness in histology applications.
Regards,
Dorothy Murphy Traczyk
National Sales Manager
Hacker Instruments & Industries Inc.
PO Box 1176
Winnsboro, SC 29180
1-800-442-2537
hackerlab <@t> aol.com
_www.hacker_ (http://www.hacker/) insruments.com
_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
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------------------------------
Message: 14
Date: Mon, 26 Sep 2005 18:03:15 -0500
From: "Nelson, Sandy - PPH" <Sandy.Nelson <@t> tenethealth.com>
Subject: [Histonet] RE: Posting for Histology List Serve
To: "Histology List Serve" <histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<DD6F44B1686B474FB72F9F55338D47D201040BE0 <@t> tenhdcthemx11.tenethealth.net>
Content-Type: text/plain; charset=iso-8859-1
> Subject: Histology Management Position
>
> A 500 bed hospital adjacent to the Houston Medical Center has an opening
for a full time Histology Supervisor. This candidate should have strong
leadership skills and prior management experience is preferred. Interested?
Call 713.527.5292.
>
> Sandra Nelson
> Director Laboratory Services
> Park Plaza Hospital
> Houston, Texas
> 713.527.5292
> sandy.nelson <@t> tenethealth.com
> ** This is not intended to be a legally binding or legally effective
signature
>
>
>
------------------------------
Message: 15
Date: Mon, 26 Sep 2005 20:27:30 -0400
From: "Andrea T. Hooper" <anh2006 <@t> med.cornell.edu>
Subject: [Histonet] Staining fibrin - thrombus
To: histonet <@t> lists.utsouthwestern.edu
Message-ID: <p06200716bf5e3ee5d5c0@[140.251.146.71]>
Content-Type: text/plain; charset=us-ascii; format=flowed
Hi All!
If you wanted to stain a paraffin section (frozen also available) of
mouse tissue and prove what you are seeing is a result of a
thrombotic event what stains would you use?
I was thinking to do an anti-fibrin immunostain (antibody suggestions
welcome) and perhaps some sort of fibrin special stain (I came up
with Carstairs' and Weigert's as probably being the best). Any
recommendations for antibodies, techniques, protocols, words of
wisdom?
Please any information, detailed or not, will be put to good use ...
and thanks!
Andrea
--
------------------------------
Message: 16
Date: Mon, 26 Sep 2005 21:09:38 -0400
From: "Katri Tuomala" <katri <@t> cogeco.ca>
Subject: [Histonet] IHC background problems on chicken embryos
To: "HistoNet Server" <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <000b01c5c300$225661f0$6a9a9618 <@t> Katri>
Content-Type: text/plain; format=flowed; charset="iso-8859-1";
reply-type=original
Hi Histonetters,
A colleague of mine is trying to get VWF (Dakocytomation) and VEGF(R&D
Systems) working on chicken tissue. The antibodies are performing well on
human and rat tissue, but excessive background is the problem with the
chicken. Would the blocking serums (normal goat and normal rabbit
respectively) be the problem as well as the biotinylated secondary reagents
raised in goat and rabbit? My experience is strictly with human tissues, so
I am stumped. To my knowledge neither of these antibodies have been proven
to work with chicken, so maybe that is the problem.
Any advice from "chicken" people?
Katri
Katri Tuomala
Hamilton, Ontario, Canada
------------------------------
Message: 17
Date: Mon, 26 Sep 2005 18:29:53 -0700
From: "Ralph Puchalski" <ralphpu <@t> alleninstitute.org>
Subject: [Histonet] Pink precipitate is monoformazan from BCIP/NBT
reaction on ISH?
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<BC3AB5FAA3FD154692EB0A6B897113F2EE1B78 <@t> AIBSMSX.corp.alleninstitute.org>
Content-Type: text/plain; charset="us-ascii"
I am trying to figure out the origin of the pink precipitate in the
image I posted on http://www.histonet.org/site_images_frame.asp
Please go to the image entitled: Pink Precipitate ver2.jpg. It is at
the top of the list on 9/26/05, posted by Ralph Puchalski. To see the
pink precipitate artifact, please open the image and set the scroll bars
on the bottom and right side of the image at their 1/2 way points. It
is ugly!
I think this precipitate is the aggregation of the monoformazan
intermediate generated from NBT (after dephosphorylation of BCIP by
alkaline phosphatase) that is not completely reduced to diformazan, the
insoluble dark blue or black precipitate that labels cells expressing
target mRNAs in our in situ hybridization reactions.
We don't know how the monoformazan adheres to the sections of tissue.
It appears to be non-covalent due to the tendency of the monoformazan to
migrate under the coverslip in the aqueous mounting medium that has yet
to dry, and form clumps or aggregates or pools as seen in the picture.
The monoformazan is soluble in ethanol so doesn't form pools or
aggregates. But as soon as it is exposed to an aqueous medium, it
precipitates.
If we mount the post-ISH tissue sections with an organic based mounting
medium like UV-CureMount (Instrumedics), the monoformazan might cause
the entire section to turn to a brown tint as seen on
http://www.histonet.org/site_images_frame.asp
Please go to the image Brown Tint 1.jpg. It is 4th image from the top
on 9/26, posted by Ralph Puchalski. The lower image is mounted with UV
CureMount, and the upper was with aqueous Hydro Matrix. There is no
pooling or precipitation of monoformazan with UV CureMount, but I think
it does cause the entire section to turn brown.
Question: How do we eliminate this problem, which I believe is
monoformazan? If we reduce it fully using ascorbate, the section (later
mounted with HyrdoMatrix) turns dark blue or purple, the same color as
our ISH signal. We have tried washing off the monoformazan with 100%
ethanol prior to coverslipping, but only small amounts are removed.
100% acetone also does not work effectively.
Please let me know if you have any ideas that might help us eliminate
this problem.
Thank you,
Ralph
Ralph Puchalski, Ph.D.
Manager, Process Engineering and Automation
Allen Institute for Brain Science
551 N. 34th Street, Suite 200
Seattle, WA 98103
ralphpu <@t> alleninstitute.org
Tel: 206-548-7041 Fax: 206-548-7071
www.brainatlas.org
------------------------------
Message: 18
Date: Mon, 26 Sep 2005 18:28:56 -0700
From: "Histology" <histology <@t> 2hosts.com>
Subject: Re: [Histonet] Microwave PROCCESSING
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <009f01c5c302$d761a9e0$c901fec0 <@t> FAMILYROOM>
Content-Type: text/plain; format=flowed; charset="iso-8859-1";
reply-type=original
We are using microwave processing. We've had to process fatty tissues, lymph
nodes and cell blocks on a standard VIP, but are using the Sakura Xpress for
the rest. We've recently had a software and reagent update which will allow
all tissues types to run on the standard 1.5 hour program. There was some
adjustment (still more to be made) with the folks that gross specimens
however. Sometimes the tissues are still a bit too thick. Our pathologists
come in between 7 and 8 am and leave mid-afternoon through early evening
depending on the individual. We haven't made any changes in IHC or Special
Stains methods. Oh, and for those that like to have their tissues tinted,
the new Sakura process allows eosin to be added to the preprocessing
solution.
Hope this helps.
Saundra
----- Original Message -----
From: "Jesus Ellin" <JEllin <@t> yumaregional.org>
To: <histonet <@t> lists.utsouthwestern.edu>
Sent: Thursday, September 22, 2005 11:15 AM
Subject: [Histonet] Microwave PROCCESSING
> Nice to see that everyone made it back from NSH and are doing well. I
> have a nice question that might start some serious discussion. Microwave
> proccessing!!!! My question is how many people out there are doing it and
> what issues did you have to work through to apply this technology to
> everyday use??? How are you handling the testing of your immuno's since
> you have to use tissue that has been run by the microwave proccessor???
> How is the transcription being done??? What time are the pathologist
> coming in and how LATE are they staying?? Are you proccessing all tissue
> type or only small biopies, breast biopsies, etc.?? What road block have
> you come up against??? It this the rule to proccess all tissue or is this
> the exception?? How are you handling reference lab consultation with your
> tissue that is proccessed on the Microwave and sending it to a lab for
> instance AFIP for consult and they only use the conventional proccessing??
> Is anyone out there doing both and how do they keep track of the different
> types of tissue that have been proccessed within the different proccessor,
> if you are indeed using both. What additional costs did you do you have,
> with there new product from the proccessor??
>
>
> Our pathologist came back and are now foaming at the mouth for this
> technology. We are not against this, but there are avenues that need to
> be addressed, especially with IHC, FISH, and overall workflow. Any help
> would greatly be appreciated and needed at this time.
>
> Your in Formalin
>
> Jesus Ellin
> Yuma Regional Medical Center
>
>
>
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
------------------------------
Message: 19
Date: Mon, 26 Sep 2005 18:31:03 -0700
From: "Histology" <histology <@t> 2hosts.com>
Subject: Re: [Histonet] Embedding centers
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <00a801c5c303$2329b020$c901fec0 <@t> FAMILYROOM>
Content-Type: text/plain; format=flowed; charset="iso-8859-1";
reply-type=original
My favorite is still the Shandon, but we have both Shandon and Leica. I
really don't like either the Leica or the Sakura models.
Saundra Ellis
Kaiser Permanente
Histology Supervisor
----- Original Message -----
From: "Snider, Deanna" <dsnider <@t> shrinenet.org>
To: <histonet <@t> lists.utsouthwestern.edu>
Sent: Thursday, September 22, 2005 11:54 AM
Subject: [Histonet] Embedding centers
> Hi everyone! I once again am in need of your knowledge and guidance! My
> existing embedding center is on the fritz. We are looking into purchasing
> a
> new one. Which brands do you recommend? Why? I am a low volume research
> lab, working with clinically engineered skin and human on mouse grafts. I
> looked at the archives but not much was there!
> Thanks in advance,
>
> Deanna Snider HT ASCP
> Shriners Hospital for Children
> Research Dept.
> Cincinnati, Oh
> 513-872-6388
>
> CONFIDENTIALITY NOTICE: This e-mail communication and any attachments may
> contain confidential and privileged information for the use of the
> designated recipients. If you are not the intended recipient, (or
> authorized to receive for the recipient) you are hereby notified that you
> have received this communication in error and that any review, disclosure,
> dissemination, distribution or copying of it or its contents is
> prohibited.
> If you have received this communication in error, please destroy all
> copies
> of this communication and any attachments and contact the sender by reply
> e-mail or telephone (813) 281-0300.
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
------------------------------
Message: 20
Date: Mon, 26 Sep 2005 19:59:41 -0700
From: Sam Vaughn <samvaughnhisto <@t> gmail.com>
Subject: [Histonet] section edges lifting
To: histonet <@t> lists.utsouthwestern.edu
Message-ID: <1582e1c05092619596afc6ce1 <@t> mail.gmail.com>
Content-Type: text/plain; charset=ISO-8859-1
I'm new to histonet and have a question about histo staining.
I have 6 um paraffin sections of mouse knee joints on uncoated superfrost
plus slides. I collect the sections from a distilled water bath and let them
drain at least 30min before placing them at 37 degrees overnight before
staining. After Safranin O/ Fast Green staining a very thin area around the
edge at the edge of the tissue lifts off the slide rendering it impossible
to visualize with the microscope. Of course this is the the articular
surface which we are interested in! I've used the same slides in the past
without any coating and it has worked fine. I'm in a new lab now and trying
to get things running. Any help you could give me would be really
appreciated!
Thanks,
Sam
------------------------------
Message: 21
Date: Tue, 27 Sep 2005 00:41:56 -0400
From: John Kiernan <jkiernan <@t> uwo.ca>
Subject: Re: [Histonet] Pink precipitate is monoformazan from BCIP/NBT
reaction onISH?
To: Ralph Puchalski <ralphpu <@t> alleninstitute.org>
Cc: histonet <@t> lists.utsouthwestern.edu
Message-ID: <4338CD94.F2BDE464 <@t> uwo.ca>
Content-Type: text/plain; charset=us-ascii
Dear Ralph,
Your email is full of abbreviations and jargon.
Am I right in thinking it's a question about
localizing alkaline phosphatase activity by an
indoxyl-tetrazolium method? If so, please provide
a reference to the original publication and let us
all know if you followed it exactly or made any
changes. Part of your email suggests that the
alkaline phosphatase activity is not endogenous
but part of an amplification system used in
in situ hybridization. The pH optima of endogenous
and label alkaline phosphatases differ.
The later paragraphs of your message indicate that
you may not fully understand the significance of
the mono- and diformazan products of reduction of
nitro-BT. Both colours result from reduction of
the tetrazolium salt, but you need controls to
prove that the reduction was by bromochloroindoxyl
and not by other reducing agents (such as diaphorases)
in the tissue. Non-enzymatic reduction of tetrazolium
salts by -SH has been a well known artifact {"nothing
dehydrogenase") for 40-45 years.
John A. Kiernan
Anatomy Dept, UWO
London, Canada.
___________________________________________________
Ralph Puchalski wrote:
>
> I am trying to figure out the origin of the pink precipitate in the
> image I posted on http://www.histonet.org/site_images_frame.asp
>
> Please go to the image entitled: Pink Precipitate ver2.jpg. It is at
> the top of the list on 9/26/05, posted by Ralph Puchalski. To see the
> pink precipitate artifact, please open the image and set the scroll bars
> on the bottom and right side of the image at their 1/2 way points. It
> is ugly!
>
> I think this precipitate is the aggregation of the monoformazan
> intermediate generated from NBT (after dephosphorylation of BCIP by
> alkaline phosphatase) that is not completely reduced to diformazan, the
> insoluble dark blue or black precipitate that labels cells expressing
> target mRNAs in our in situ hybridization reactions.
>
> We don't know how the monoformazan adheres to the sections of tissue.
> It appears to be non-covalent due to the tendency of the monoformazan to
> migrate under the coverslip in the aqueous mounting medium that has yet
> to dry, and form clumps or aggregates or pools as seen in the picture.
> The monoformazan is soluble in ethanol so doesn't form pools or
> aggregates. But as soon as it is exposed to an aqueous medium, it
> precipitates.
>
> If we mount the post-ISH tissue sections with an organic based mounting
> medium like UV-CureMount (Instrumedics), the monoformazan might cause
> the entire section to turn to a brown tint as seen on
> http://www.histonet.org/site_images_frame.asp
>
> Please go to the image Brown Tint 1.jpg. It is 4th image from the top
> on 9/26, posted by Ralph Puchalski. The lower image is mounted with UV
> CureMount, and the upper was with aqueous Hydro Matrix. There is no
> pooling or precipitation of monoformazan with UV CureMount, but I think
> it does cause the entire section to turn brown.
>
> Question: How do we eliminate this problem, which I believe is
> monoformazan? If we reduce it fully using ascorbate, the section (later
> mounted with HyrdoMatrix) turns dark blue or purple, the same color as
> our ISH signal. We have tried washing off the monoformazan with 100%
> ethanol prior to coverslipping, but only small amounts are removed.
> 100% acetone also does not work effectively.
>
> Please let me know if you have any ideas that might help us eliminate
> this problem.
>
> Thank you,
>
> Ralph
>
> Ralph Puchalski, Ph.D.
> Manager, Process Engineering and Automation
> Allen Institute for Brain Science
> 551 N. 34th Street, Suite 200
> Seattle, WA 98103
>
> ralphpu <@t> alleninstitute.org
> Tel: 206-548-7041 Fax: 206-548-7071
> www.brainatlas.org
>
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