[Histonet] Collagen stain
Dear Dr. Kiernan and Barbara, and colleagues,
I am also very interested in collagen staining using blue dyes for better contrast for image analysis. For now we use picro-sirius red overnight without counterstaining. Could you send me detailed protocol, please.I mean: time of incubation, wash, differentiation ect.As well could you give me a name of Dr. Kiernan book and where I can buy it.
Thank you at advance.
Galina Deyneko
Novartis Cambridge MA
617-871-7613
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Today's Topics:
1. Re: removing excess x-gal stain (LONG) (John Kiernan)
2. bx sponges VS no bx sponges (Snider, Deanna)
3. Appropriate cassette types (eileen.lonergan@verizon.net)
4. RE: Polioma virus - antibody (Drew Sally A.)
5. Re: staining collagen (Barbara Bublava)
6. beta-oestrogen receptor (Edwards, R.E.)
7. cryosette (Kim Merriam)
8. RE: cryosette (Alan Bright)
9. Re: staining collagen (John A. Kiernan)
10. RE: Appropriate cassette types (Bonner, Janet)
11. H&E staining (eileen dusek)
12. RE: Appropriate cassette types (Kemlo Rogerson)
13. Re: H&E staining (Rene J Buesa)
14. RE: Polioma virus - antibody (Joanne Mauger)
15. Re: H&E staining (Massimo)
16. RE: H&E staining (Malcolm McCallum)
17. RE: H&E staining (James Watson)
18. Uneven Staining (Mary Parker)
----------------------------------------------------------------------
Message: 1
Date: Mon, 27 Feb 2006 01:26:41 -0500
From: John Kiernan
Subject: Re: [Histonet] removing excess x-gal stain (LONG)
To: Linda K Bauer
Cc: histonet
Message-ID: <44029BA1.131D6E00@uwo.ca>
Content-Type: text/plain; charset=us-ascii
Dear Linda K Bauer,
Nobody has replied to you on Histonet for about
a week, so a few speculative comments from a
non-user of the X-gal technique may be better
than nothing at all. This reply has three parts.
1. How can you have "excess nonspecific X-gal
stain"? The substrate (X-gal) is hydrolysed by
beta-galactosidase and one of the products of
hydolysis, "X" (which is short for
5-bromo-4-chloroindoxyl), is rapidly oxidized (by
the ferricyanide ions in the incubation medium) to
an indigoid dye that rejoices in the name of
5,5'-dibromo-4,4'-dichloroindigo. This particular
indigoid dye sticks strongly to the nearest
protein molecules and does not diffuse into fat or
other lipids, and does not form visible crystals.
This is one of the most thoroughly studied
reactions in enzyme histochemistry. If the method
was carried out correctly there should be no
"nonspecific" deposition of
5,5'-dibromo-4,4'-dichloroindigo more than 500nm
from a beta-galactosidase enzyme molecule.
There are some minor controversies about the
sensitivity and specificity of indigogenic methods
for lysosomal enzymes (with acidic pH optima), and
for many of these enzymes there are alternative
histochemical methods.
2. I'm assuming that you are using the indigogenic
X-gal method to detect the lac-Z reporter gene,
which encodes a bacterial beta-galactosidase that
works at pH 7+. This gene is tagged onto genes
that encode more interesting proteins in
experiments with altered cell lines, transgenic
mice etc. Cells that transcribe and translate an
added or modified gene also make the bacterial
enzyme encoded by the reporter gene. Histochemical
detection of a beta-galactosidase active at ph 7+
indicates that the "more interesting proteins" are
probably being made in the same cell.
The problem with lysosomal enzymes referred to
at the end of Part 1 (above) does not arise with
indigogenic detection of the lac-Z gene product
because the pH of the medium is significantly
higher than the pH optima of lysosomal
glycosidases.
"Normal" (animal) beta-galactosidase activity
would be detected only after an unduly prolonged
incubation. The false-positive result would also
show up in your negative control sections of
tissues known not to express the lac-Z gene.
If your known-negative controls show no
blue-green colour and your transgenics (or others)
show "excess nonspecific X-gal stain" there is no
"excess". You must be working with a tissue whose
cells are putting out lots of the bacterial
enzyme. The genetic engineer should thank you for
providing evidence that the introduced genes
entered the host organism and were successfully
transcribed and translated.
If you have no known-negative and
known-positive control results, ask your boss
for more detailed advice.
3. This is an answer to your beautifully concise
two-line question, cited below. The indigoid dye
is insoluble. It was probably deposited at the
site of the enzyme, if the histochemical method
was done by the book.
John Kiernan
Anatomy, UWO
London, Canada
----------------------------------------
Linda K Bauer wrote:
>
> I would like to remove excess nonspecific x-gal stain from whole mount
> murine embryos. Does anyone have a suggestion or technique for this?
>
> Linda(Lin)Bauer
> Department of Genetics, Cell Biology, and Anatomy
> 985455 Nebraska Medical Center
> Omaha, NE 68198-5455
>
> Phone: (402) 559-2863
> Fax: (402) 559-4001
> Email: lkbauer@unmc.edu
>
> _______________________________________________
> Histonet mailing list
> Histonet@lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
------------------------------
Message: 2
Date: Mon, 27 Feb 2006 08:00:58 -0500
From: "Snider, Deanna"
Subject: [Histonet] bx sponges VS no bx sponges
To:
Message-ID:
<84BE46B37B314D409C5A17B7BAB022D666ACFE@IDC-EX-VS01.shriners.cc>
Content-Type: text/plain; charset="us-ascii"
I too have found this to be true with bx sponges also. I work with
clinically engineered skin, which is much thinner than normal human
skin. The specimens had to be kept flat. The sponges created all kinds
issues. I ran across a product, called a bx capsule, which is a mesh
screen that folds shut(like the bx cassettes). The tissue is held
securely, and flat, and the carryover is decreased dramatically and
there is no artifact. You can order these from several vendors.
Shriners Hospital for Children
Research Dept.
Cincinnatti, Oh
513-872-6388
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------------------------------
Message: 3
Date: Mon, 27 Feb 2006 07:21:25 -0600 (CST)
From: eileen.lonergan@verizon.net
Subject: [Histonet] Appropriate cassette types
To: histonet@lists.utsouthwestern.edu
Message-ID:
<25625309.1141046485781.JavaMail.root@vms064.mailsrvcs.net>
Content-Type: text/plain; charset=ISO-8859-1
Fellow Histonetters,
We are a large reference lab that processes various medical facilities tissues. We have a new client that insists on using the small screened biopsy cassettes for every tissue type, including breast, uterus, parotid gland etc.
We have ordered the appropriate routine cassettes with the slots for their large tissue but they refuse to use them and the quality of the resulting samples is sub-optimal at best.
Our patholgoist has volunteered to speak to them since most of Histology's requests fall on deaf ears.
Are there any articles and/or opinions on this issue? I have been in the field for quite a few years and this is the first time I have encountered resistance like this. Most of the grossing is done by a PA who actually does a nice job with the actual grossing, it's just using this screened cassette that we are unable to change.
Thanks for any opinions here.
Eileen Lonergan HT(ASCP)
Eileen Lonergan
(207) 749-9617 cell
(207) 324-6468 home
------------------------------
Message: 4
Date: Mon, 27 Feb 2006 07:30:14 -0600
From: "Drew Sally A."
Subject: RE: [Histonet] Polioma virus - antibody
To: "Histonet"
Message-ID:
Content-Type: text/plain; charset="US-ASCII"
We use the BK/SV40-T antibody from Calbiochem on our FFPE renal biopies
and it works well.
Sally Ann Drew, MT(ASCP)
IHC/ISH Laboratory
University of Wisconsin Hosp. & Clinics
Madison, WI 53792
(608)265-6596
-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu
[mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of Gudrun
Lang
Sent: Saturday, February 25, 2006 1:55 AM
To: Histonetliste (Histonetliste)
Subject: [Histonet] Polioma virus - antibody
Please, can somebody recommend an antibody against Polioma virus, that
works on FFPE tissue? We have the Bechmark XT, Ventana, and want to use
it on renal biopsies.
Thanks in advance
Gudrun Lang
Linz, Austria
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------------------------------
Message: 5
Date: Mon, 27 Feb 2006 14:31:01 +0100
From: "Barbara Bublava"
Subject: Re: [Histonet] staining collagen
To: "Marc Tjwa" ,
Message-ID: <015d01c63ba2$0ebff640$47a9abc1@GERICHTS9XOZZ8>
Content-Type: text/plain; charset="iso-8859-1"
hi Marc,
pikro-aniline blue:
add 0,5g Anilinblue or methylenblue to 500ml saturated aquous solution of
picric acid.
everything else is done like van Gieson
Result: collagen, basement membranes and reticulin blue; cytoplasm yellow
John Kiernanīs book says reticulin and basement membranes are not stained if
you use indigocarmine (0,5 g in 200ml) collagen should be blue-green in this
case
greetings
Barbara
----- Original Message -----
From: "Marc Tjwa"
To: "Barbara Bublava"
Sent: Sunday, February 26, 2006 4:32 PM
Subject: Re: [Histonet] staining collagen
Dear Barbara
I am also interested in the protocol that you
have suggested. Would you please be so kind to
send me the protocol by mail?
Tnx in advance
YS
Marc Tjwa
>You could use anilineblue instead of acid fuchsin. makes better contrast
>and is described in John Kiernans book
>
>tell me if you canīt find a protocol
>
>greetings
>Barbara, Vienna
>----- Original Message -----
>From: "Lobke De Bels"
>To:
>Sent: Friday, February 24, 2006 8:58 AM
>Subject: [Histonet] staining collagen
>
>
>
>Von Gieson gives a good contrast for the human eye, but the computer can't
>make
>a difference between yellow/orange and red.
>If I only use acid fushine (or aniline) everything stains pink(blue).
>
>I have to find a way to stain the connective tissue so the computer can
>clearly
>make a difference between connective tissue and the other tissue.
>
>The ideal should be a method that only stains collagen.
>
>
>Many thanks in advance for any help
>
>
>
>Lobke De Bels
>Department of Morphology
>Faculty of Veterinary Medicine
>Ghent University
>Salisburylaan 133
>9820 Merelbeke
>Belgium
>lobke.debels@ugent.be
>
--
============================================================
Marc Tjwa, MD
Center for Transgene Technology and Gene Therapy (CTG)
Flanders Interuniversity Institute for Biotechnology (VIB)
University of Leuven (KUL)
Campus Gasthuisberg, Herestraat 49
B-3000, Leuven, Belgium
Tel: +32-16-345775; Fax: +32-16-345990
E-mail: Marc.Tjwa@med.kuleuven.be
============================================================
Disclaimer: http://www.kuleuven.be/cwis/email_disclaimer.htm
------------------------------
Message: 6
Date: Mon, 27 Feb 2006 14:08:26 -0000
From: "Edwards, R.E."
Subject: [Histonet] beta-oestrogen receptor
To:
Message-ID:
Content-Type: text/plain; charset="iso-8859-1"
Looking for an antibody to beta-oestrogen receptor that works on formalin-fixed paraffin processed tissues of mice, have tried the NOVACASTRA antibody, but no luck to date.
Thanks
Richard Edwards
MRC TOXICOLOGY UNIT
LEICESTER. U.K....
------------------------------
Message: 7
Date: Mon, 27 Feb 2006 06:45:30 -0800 (PST)
From: Kim Merriam
Subject: [Histonet] cryosette
To: Histonet
Message-ID: <20060227144530.24877.qmail@web50313.mail.yahoo.com>
Content-Type: text/plain; charset=iso-8859-1
Hello all,
I received some OCT blocks from an outside vendor and they are embedded in something called a "cryosette". They are really cool. Did a google search for them and nothing came up (how often does that happen?). Anyway - does anyone know who makes these and where I can buy them?
Thanks,
Kim
Kim Merriam
Novartis
Cambridge, MA
---------------------------------
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------------------------------
Message: 8
Date: Mon, 27 Feb 2006 15:18:39 -0000
From: "Alan Bright"
Subject: RE: [Histonet] cryosette
To: "Kim Merriam"
Cc: histonet@lists.utsouthwestern.edu
Message-ID:
Content-Type: text/plain; charset="us-ascii"
Dear Kim,
We manufacture a range, what sizes are you interested in.
Best Regards
Alan Bright
Bright Instrument Co.Ltd.
St Margaret's Way
Huntingdon
Cambridgeshire
PE29 6EU
England
Tel No:+44 (0)1480 454528
Fax No:+44 (0)1480 456031
Email: abright@brightinstruments.com
Web Site: www.brightinstruments.com
-----Original Message-----
From: Kim Merriam [mailto:kmerriam2003@yahoo.com]
Sent: 27 February 2006 14:46
To: Histonet
Subject: [Histonet] cryosette
Hello all,
I received some OCT blocks from an outside vendor and they are
embedded in something called a "cryosette". They are really cool. Did
a google search for them and nothing came up (how often does that
happen?). Anyway - does anyone know who makes these and where I can buy
them?
Thanks,
Kim
Kim Merriam
Novartis
Cambridge, MA
---------------------------------
Yahoo! Mail
Bring photos to life! New PhotoMail makes sharing a breeze.
_______________________________________________
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http://lists.utsouthwestern.edu/mailman/listinfo/histonet
------------------------------
Message: 9
Date: Mon, 27 Feb 2006 10:59:42 -0500
From: "John A. Kiernan"
Subject: Re: [Histonet] staining collagen
To: Barbara Bublava
Cc: histonet@lists.utsouthwestern.edu, Marc Tjwa
Message-ID: <440321EE.4B966435@uwo.ca>
Content-Type: text/plain; charset=iso-8859-1
Methyl blue, NOT methylene blue! Methyl blue is
one of the components of aniline blue, which is
often a mixture of related anionic
triphenylmethane dyes. (Methylene blue is a
cationic thiazine dye with very different staining
capabilities.) The picro-aniline blue methods pick
up thin collagen fibres that are missed by van
Gieson. The staining pattern is closely similar to
picro-sirius red F3B, but aniline blue does not
enhance the birefringence of type 1 collagen.
John Kiernan
London, Canada.
----------------------------------------
Barbara Bublava wrote:
>
> hi Marc,
>
> pikro-aniline blue:
>
> add 0,5g Anilinblue or methylenblue to 500ml saturated aquous solution of
> picric acid.
> everything else is done like van Gieson
> Result: collagen, basement membranes and reticulin blue; cytoplasm yellow
>
> John Kiernanīs book says reticulin and basement membranes are not stained if
> you use indigocarmine (0,5 g in 200ml) collagen should be blue-green in this
> case
>
> greetings
> Barbara
>
> ----- Original Message -----
> From: "Marc Tjwa"
> To: "Barbara Bublava"
> Sent: Sunday, February 26, 2006 4:32 PM
> Subject: Re: [Histonet] staining collagen
>
> Dear Barbara
>
> I am also interested in the protocol that you
> have suggested. Would you please be so kind to
> send me the protocol by mail?
>
> Tnx in advance
>
> YS
>
> Marc Tjwa
>
> >You could use anilineblue instead of acid fuchsin. makes better contrast
> >and is described in John Kiernans book
> >
> >tell me if you canīt find a protocol
> >
> >greetings
> >Barbara, Vienna
> >----- Original Message -----
> >From: "Lobke De Bels"
> >To:
> >Sent: Friday, February 24, 2006 8:58 AM
> >Subject: [Histonet] staining collagen
> >
> >
> >
> >Von Gieson gives a good contrast for the human eye, but the computer can't
> >make
> >a difference between yellow/orange and red.
> >If I only use acid fushine (or aniline) everything stains pink(blue).
> >
> >I have to find a way to stain the connective tissue so the computer can
> >clearly
> >make a difference between connective tissue and the other tissue.
> >
> >The ideal should be a method that only stains collagen.
> >
> >
> >Many thanks in advance for any help
> >
> >
> >
> >Lobke De Bels
> >Department of Morphology
> >Faculty of Veterinary Medicine
> >Ghent University
> >Salisburylaan 133
> >9820 Merelbeke
> >Belgium
> >lobke.debels@ugent.be
> >
>
> --
> ============================================================
> Marc Tjwa, MD
> Center for Transgene Technology and Gene Therapy (CTG)
> Flanders Interuniversity Institute for Biotechnology (VIB)
> University of Leuven (KUL)
> Campus Gasthuisberg, Herestraat 49
> B-3000, Leuven, Belgium
> Tel: +32-16-345775; Fax: +32-16-345990
> E-mail: Marc.Tjwa@med.kuleuven.be
> ============================================================
------------------------------
Message: 10
Date: Mon, 27 Feb 2006 11:25:02 -0500
From: "Bonner, Janet"
Subject: RE: [Histonet] Appropriate cassette types
To: eileen.lonergan@verizon.net, histonet@lists.utsouthwestern.edu
Message-ID:
Content-Type: text/plain; charset=iso-8859-1
Perhaps if you copy out your letter and send it with the next batch of poorly processed tissue.......
________________________________
From: histonet-bounces@lists.utsouthwestern.edu on behalf of eileen.lonergan@verizon.net
Sent: Mon 2/27/2006 8:21 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Appropriate cassette types
Fellow Histonetters,
We are a large reference lab that processes various medical facilities tissues. We have a new client that insists on using the small screened biopsy cassettes for every tissue type, including breast, uterus, parotid gland etc.
We have ordered the appropriate routine cassettes with the slots for their large tissue but they refuse to use them and the quality of the resulting samples is sub-optimal at best.
Our patholgoist has volunteered to speak to them since most of Histology's requests fall on deaf ears.
Are there any articles and/or opinions on this issue? I have been in the field for quite a few years and this is the first time I have encountered resistance like this. Most of the grossing is done by a PA who actually does a nice job with the actual grossing, it's just using this screened cassette that we are unable to change.
Thanks for any opinions here.
Eileen Lonergan HT(ASCP)
Eileen Lonergan
(207) 749-9617 cell
(207) 324-6468 home
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------------------------------
Message: 11
Date: Mon, 27 Feb 2006 08:30:48 -0800 (PST)
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