[Histonet] RE: Histonet Digest, Vol 117, Issue 20

Wed Aug 21 07:42:06 CDT 2013

John,
Here is a link to a Picric Acid Free Bouins:
http://www.americanmastertech.com/store/main.aspx?p=ItemDetailStyles&item=FXBOU2100
We found that it fixed very well, and preserved nuclear detail better than "real" Bouins over extended fixation time.  However, it did not decal like Bouin's does.
-Liz

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of john <@t> imebinc.com
Sent: Tuesday, August 20, 2013 5:37 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] RE: Histonet Digest, Vol 117, Issue 20

Hi Histonet,
 I am interested in a Bouins Substitute to eliminate the use of Picric Acid, and limit the use of extreme percentage of formalin. I have heard of Platinum Chloride and Zinc Chloride as a possible replacement to the original formual,any input is appreciated. 

Thank You
John/Eco safe alternative

-----Original Message-----
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Subject: Histonet Digest, Vol 117, Issue 20

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Today's Topics:

   1. Re: Histonet Digest, Vol 117, Issue 19 (Mesru T)
   2. AUTO: Bruce Palmatier is out of the office (returning
      08/19/2013) (Bruce_Palmatier <@t> vwr.com)
   3. EDTA decal (Teri Johnson)
   4. post-fixation for Mallory Trichrome (Rui TAHARA)
   5. AW: [Histonet] post-fixation for Mallory Trichrome (Gudrun Lang)
   6. AW: [Histonet] EDTA decal (Gudrun Lang)
   7. edge artefact on core needle biopsy (Gudrun Lang)
   8. Cryostat cleaning (Cynthia Baranowski)
   9. RE: IHC after EDTA decalcifying (pruegg <@t> ihctech.net)
  10. RE: EDTA decal (pruegg <@t> ihctech.net)

----------------------------------------------------------------------

Message: 1
Date: Fri, 16 Aug 2013 13:29:30 -0400
From: Mesru T <turkekul <@t> gmail.com>
Subject: [Histonet] Re: Histonet Digest, Vol 117, Issue 19
To: histonet <@t> lists.utsouthwestern.edu
Message-ID:
	<CACf3QnRuLdSxZuOy6mO1phn18LaMj4vjbVRK7ah-mQnXwBDU4A <@t> mail.gmail.com>
Content-Type: text/plain; charset=ISO-8859-1

Hi Collen,

You had great responses to your question. I do stain mouse femurs for IHC and IF. We fix in NBF overnight and decal for 4-5 days in 0.5M EDTA pH=8 at room temp. After decal we wash with running tap water and re-fix for 4 hrs with NBF. 90% of the antibodies we use on undecal tissues worked on the decal femurs.

Mesru

------------------------------

Message: 2
Date: Fri, 16 Aug 2013 13:51:33 -0400
From: Bruce_Palmatier <@t> vwr.com
Subject: [Histonet] AUTO: Bruce Palmatier is out of the office
	(returning	08/19/2013)
To: histonet <@t> lists.utsouthwestern.edu
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	<OF25D46F01.CF4FEDA7-ON85257BC9.00621AC1-85257BC9.00621AC1 <@t> vwr.com>
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I am out of the office until 08/19/2013.

I will be out of the office today. If you have a question about an order or need assistance placing an order, or if the matter is urgent, please call
877-881-1192 between 8am and 8pm and a VWR Healthcare Service associate will assist you. For quote requests and pricing-related inquiries, I will respond with 24 hours.

Thank You,

Bruce Palmatier
VWR Healthcare
mobile 484-319-5563

Note: This is an automated response to your message  "Histonet Digest, Vol 117, Issue 19" sent on 8/16/2013 1:04:20 PM.

This is the only notification you will receive while this person is away.

------------------------------

Message: 3
Date: Fri, 16 Aug 2013 17:54:03 +0000
From: Teri Johnson <TJohnson <@t> gnf.org>
Subject: [Histonet] EDTA decal
To: "'cforster <@t> umn.edu'" <cforster <@t> umn.edu>
Cc: "histonet <@t> lists.utsouthwestern.edu"
	<histonet <@t> lists.utsouthwestern.edu>
Message-ID: <9F3CFEE76E51B64991C7485270890B40497B2C12 <@t> EX5.lj.gnf.org>
Content-Type: text/plain; charset="us-ascii"

Hi Colleen,

I would say it's unusual, but not completely impossible that EDTA has interfered with your IHC.  We had that problem with demonstrating B-galactosidase in mouse bones. If we decalcified it in EDTA after whole mount staining in X-gal, the blue staining was removed. But if we decalcified it in formic acid, the stain was retained.

I think a few years ago, Biogenex had a room temperature antigen retrieval solution for acid decalcified bone (not the same as your situation, I know).
But I think it was largely an alkaline solution you let the slides sit for maybe 30 minutes prior to staining. Might be worthwhile to try a different retrieval method for these and see what happens.

Otherwise, are you sure you are using a clone that reacts in mouse tissue?
We use Rabbit monoclonal SP6.

Teri Johnson
Manager, Histology
GNF - San Diego, CA
858-332-4752

------------------------------

Message: 4
Date: Sat, 17 Aug 2013 06:32:50 +0900
From: Rui TAHARA <ruio7 <@t> hotmail.com>
Subject: [Histonet] post-fixation for Mallory Trichrome
To: "histonet <@t> lists.utsouthwestern.edu"
	<histonet <@t> lists.utsouthwestern.edu>
Message-ID: <BAY171-W131137449A963C7A68A258F9470 <@t> phx.gbl>
Content-Type: text/plain; charset="iso-2022-jp"

Hello, 

There seems to be a lot of suggestions for Mallory Trichrome staining involved in Acid fusin, Aniline blue, and orange G.

My sample of avian embryos were
fixed with Formalin based fixative (4% paraformaldehyde in phosphate-buffered saline and 1% glutaraldehyde) overnight, in case of late embryos bones were decalcified, and followed standard  alcohol series, paraffin embedded, and 10 micron sectioned. 
The slides were dehydrated,
stained with 0.5 % Acid fushin for 10min, then without wash, 0.5% Aniline blue and 2 % orange G in 1% phosphomolybdic acid solution for 20  min, then undergone ethanol series, cleared and mounted.

Now I know that formalin fixed
sample do not present optimized trichrome colors based on your websites and references. Yet the stained sections of late stage embryo do still show near optimized colors whereas those of  early stage embryo show almost no blue or very dark blue, almost gray color for cartilages and most of morphologies as purple-redish colors.

Since I tested staining on late
embryos first I thought staining would work on early embryos. Does anyone provide me explanation why the staining mostly shows red-ish color and not optimized color for cartilage in early  embryos? Is that because of the formalin-fixed embryos although late stage embryos fixed with formalin still show the blue for cartilage?

Another question is related to
fixative. I prefer not to use bousin fixative due to picric acid and it seems that Citrate buffer or Gram$B!G(Bs iodine solution can be substituted to bousin post-fixative. Have anyone tried  these solution for Mallory Trichrome? Do those methods show near optimized color for Mallory trichrome?

Any suggestion is appreciated. 
Thank you, 

Rui TAHARA
PhD candidate
McGill University, Biology Department

------------------------------

Message: 5
Date: Sat, 17 Aug 2013 09:26:38 +0200
From: "Gudrun Lang" <gu.lang <@t> gmx.at>
Subject: AW: [Histonet] post-fixation for Mallory Trichrome
To: "'Rui TAHARA'" <ruio7 <@t> hotmail.com>
Cc: histonet <@t> lists.utsouthwestern.edu
Message-ID: <000c01ce9b1b$1ca76dc0$55f64940$@gmx.at>
Content-Type: text/plain;	charset="iso-8859-1"

Hi,
I think staining would be improved, when you use Bouin as postfixative ( 1h hour, 600C).
Try to invite a further step between Fuchsin and Anilinblue. When you incubate the slides in 1% phosphomolybdic acid for 5-10 min, you can see decolorization of cardilage. This improves later on anilin-binding.
You may control the differentiation-step under microscope. Anilinblue can be reduced to 5 min afterwards.

You may also consider, that there's a real difference in cardilage-architecture of the early and late samples, that takes influence on staining-abilities.

Gudrun Lang

-----Urspr|ngliche Nachricht-----
Von: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] Im Auftrag von Rui TAHARA
Gesendet: Freitag, 16. August 2013 23:33
An: histonet <@t> lists.utsouthwestern.edu
Betreff: [Histonet] post-fixation for Mallory Trichrome

Hello, 

There seems to be a lot of suggestions for Mallory Trichrome staining involved in Acid fusin, Aniline blue, and orange G.

My sample of avian embryos were
fixed with Formalin based fixative (4% paraformaldehyde in phosphate-buffered saline and 1% glutaraldehyde) overnight, in case of late embryos bones were decalcified, and followed standard  alcohol series, paraffin embedded, and 10 micron sectioned. 
The slides were dehydrated,
stained with 0.5 % Acid fushin for 10min, then without wash, 0.5% Aniline blue and 2 % orange G in 1% phosphomolybdic acid solution for 20  min, then undergone ethanol series, cleared and mounted.

Now I know that formalin fixed
sample do not present optimized trichrome colors based on your websites and references. Yet the stained sections of late stage embryo do still show near optimized colors whereas those of  early stage embryo show almost no blue or very dark blue, almost gray color for cartilages and most of morphologies as purple-redish colors.

Since I tested staining on late
embryos first I thought staining would work on early embryos. Does anyone provide me explanation why the staining mostly shows red-ish color and not optimized color for cartilage in early  embryos? Is that because of the formalin-fixed embryos although late stage embryos fixed with formalin still show the blue for cartilage?

Another question is related to
fixative. I prefer not to use bousin fixative due to picric acid and it seems that Citrate buffer or Grams iodine solution can be substituted to bousin post-fixative. Have anyone tried  these solution for Mallory Trichrome? Do those methods show near optimized color for Mallory trichrome?

Any suggestion is appreciated. 
Thank you, 

Rui TAHARA
PhD candidate
McGill University, Biology Department

------------------------------

Message: 6
Date: Sat, 17 Aug 2013 10:08:29 +0200
From: "Gudrun Lang" <gu.lang <@t> gmx.at>
Subject: AW: [Histonet] EDTA decal
To: "'Teri Johnson'" <TJohnson <@t> gnf.org>
Cc: histonet <@t> lists.utsouthwestern.edu
Message-ID: <000d01ce9b20$f5dd1a90$e1974fb0$@gmx.at>
Content-Type: text/plain;	charset="iso-8859-1"

We experienced different IHC staining qualities on bone marrow, that was decalcified with EDTA or formic acid. I remember, our CD38 antibody was always very weak after EDTA and made no problems after formic acid.
I think it is very difficult to make an over-all-statement for all antigens/all antibodies. 
Gudrun 

-----Urspr|ngliche Nachricht-----
Von: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] Im Auftrag von Teri Johnson
Gesendet: Freitag, 16. August 2013 19:54
An: 'cforster <@t> umn.edu'
Cc: histonet <@t> lists.utsouthwestern.edu
Betreff: [Histonet] EDTA decal

Hi Colleen,

I would say it's unusual, but not completely impossible that EDTA has interfered with your IHC.  We had that problem with demonstrating B-galactosidase in mouse bones. If we decalcified it in EDTA after whole mount staining in X-gal, the blue staining was removed. But if we decalcified it in formic acid, the stain was retained.

I think a few years ago, Biogenex had a room temperature antigen retrieval solution for acid decalcified bone (not the same as your situation, I know).
But I think it was largely an alkaline solution you let the slides sit for maybe 30 minutes prior to staining. Might be worthwhile to try a different retrieval method for these and see what happens.

Otherwise, are you sure you are using a clone that reacts in mouse tissue?
We use Rabbit monoclonal SP6.

Teri Johnson
Manager, Histology
GNF - San Diego, CA
858-332-4752

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------------------------------

Message: 7
Date: Sat, 17 Aug 2013 11:18:40 +0200
From: "Gudrun Lang" <gu.lang <@t> gmx.at>
Subject: [Histonet] edge artefact on core needle biopsy
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <000001ce9b2a$c3397f20$49ac7d60$@gmx.at>
Content-Type: text/plain;	charset="us-ascii"

Hi all!

We had a discussion on the cause of IHC positivity/false-positivity on the edges of core needle biopsies. (eg. breast, ER)

One said, it is due to overfixation with formaldehyde-concentrations above 5%. (=false pos.)

My opinion is, that's it's rather an effect of underfixation in the center in those cases. (= real pos.)

In my lab, we rarely see this margin-effect and the biopsies are fixed for 6 hours at least.

I searched in Pubmed for publications on this assay, but without success.

Can anyone help me out with any literature? Or personal experience?

Thanks in advance

Gudrun Lang

Ltd. BMA Histolab Linz, Austria

------------------------------

Message: 8
Date: Sat, 17 Aug 2013 08:04:24 -0400
From: Cynthia Baranowski <clb1158 <@t> yahoo.com>
Subject: [Histonet] Cryostat cleaning
To: "Histonet <@t> lists.utsouthwestern.edu"
	<Histonet <@t> lists.utsouthwestern.edu>
Message-ID: <7A5721EF-A354-493C-BCB1-FFE4FE5E3597 <@t> yahoo.com>
Content-Type: text/plain;	charset=us-ascii

The only method acceptable to CAP is bringing the cryostat to room temp and disinfecting weekly UV light will not be an acceptable alternative by CAP.
We clean out all tissue debris, wipe down with abs alcohol, bring cryostat to room temp, use Cavicide to disinfect, wipe down again with abs alcohol.
We use 70 % alcohol for daily cleaning.
Cindy Baranowski
Emory Healthare
Atlanta, GA

Sent from my iPad

------------------------------

Message: 9
Date: Sat, 17 Aug 2013 10:39:03 -0600
From: <pruegg <@t> ihctech.net>
Subject: RE: [Histonet] IHC after EDTA decalcifying
To: "'Colleen Forster'" <cforster <@t> umn.edu>,	"'Histonet'"
	<histonet <@t> lists.utsouthwestern.edu>
Message-ID: <003f01ce9b68$498e2700$dcaa7500$@ihctech.net>
Content-Type: text/plain;	charset="iso-8859-1"

Colleen,

The antigen expression you are getting in the human tonsil may not be expressed in the mouse femur, plus u have to make sure the ab u are using cross reacts with mouse tissue, if it is anti human it may or may not react in mouse, the ab data sheet should indicate species reactivity.  The edta should not be the problem here.

Patsy

Patsy Ruegg, HT(ASCP)QIHC
Ruegg IHC Consulting
40864 E. Arkansas Ave
Bennett, CO 80102
H 303-644-4538
C 720-281-5406
pruegghm <@t> hotmail.com
rueggihcconsultingpr <@t> outlook.com

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: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Colleen Forster
Sent: Thursday, August 15, 2013 5:59 PM
To: Histonet
Subject: [Histonet] IHC after EDTA decalcifying

Hello fellow histonetters,

Can someone who has worked with decalcifyied bone tell me if EDTA interferes with IHC staining? I was under the impression it did not but cannot get staining in mouse femurs that I have decaled in EDTA. I have used both the steamer and the decloaker for retrieval and in the human tonsil the stain is great.

Any suggestions......

Thanks in advance!

Colleen Forster
U of MN

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------------------------------

Message: 10
Date: Sat, 17 Aug 2013 10:42:08 -0600
From: <pruegg <@t> ihctech.net>
Subject: RE: [Histonet] EDTA decal
To: "'Teri Johnson'" <TJohnson <@t> gnf.org>,	<cforster <@t> umn.edu>
Cc: histonet <@t> lists.utsouthwestern.edu
Message-ID: <004001ce9b68$b7784660$2668d320$@ihctech.net>
Content-Type: text/plain;	charset="iso-8859-1"

Good idea.  I have to soak the edta decaled tissues in an alkaline solution inorder to restore enzyme histochemical staining for TRAP, might be the same issue for some IHC.

Patsy Ruegg, HT(ASCP)QIHC
Ruegg IHC Consulting
40864 E. Arkansas Ave
Bennett, CO 80102
H 303-644-4538
C 720-281-5406
pruegghm <@t> hotmail.com
rueggihcconsultingpr <@t> outlook.com

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: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Teri Johnson
Sent: Friday, August 16, 2013 11:54 AM
To: 'cforster <@t> umn.edu'
Cc: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] EDTA decal

Hi Colleen,

I would say it's unusual, but not completely impossible that EDTA has
interfered with your IHC.  We had that problem with demonstrating
B-galactosidase in mouse bones. If we decalcified it in EDTA after whole
mount staining in X-gal, the blue staining was removed. But if we
decalcified it in formic acid, the stain was retained.

I think a few years ago, Biogenex had a room temperature antigen retrieval
solution for acid decalcified bone (not the same as your situation, I know).
But I think it was largely an alkaline solution you let the slides sit for
maybe 30 minutes prior to staining. Might be worthwhile to try a different
retrieval method for these and see what happens.

Otherwise, are you sure you are using a clone that reacts in mouse tissue?
We use Rabbit monoclonal SP6.

Teri Johnson
Manager, Histology
GNF - San Diego, CA
858-332-4752

_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

------------------------------

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End of Histonet Digest, Vol 117, Issue 20
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