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Today's Topics:
1. Van Gieson question.... (AGrobe2555 <@t> aol.com)
2. Re: Van Gieson question.... (Akemi Allison-Tacha)
3. Re: what do people for ISH (Mikael Niku)
4. BUFFER (Valeria Berno)
5. Pathology Cost Analysis (Amy Self)
6. Shopping for histology item (Jackie M O'Connor)
7. Re: what do people for ISH-depc (Emily Sours)
8. AW: [Histonet] Van Gieson question.... (Gudrun Lang)
9. RE: Shopping for histology item/Jackie (Joyce Cline)
----------------------------------------------------------------------
Message: 1
Date: Thu, 5 Jul 2007 16:55:46 EDT
From: AGrobe2555 <@t> aol.com
Subject: [Histonet] Van Gieson question....
To: histonet <@t> lists.utsouthwestern.edu
Message-ID: <d64.b8c1d18.33beb4d2 <@t> aol.com>
Content-Type: text/plain; charset="US-ASCII"
Good afternoon,
We are doing an Elastic Van Gieson stain on FFPE tissues and have come up
against a bit of a question. While the elastin seems to be staining quite
nicely, the nuclei either are very faintly stained or not at all. We have
shortened the differentiation time in the FeCl, with little difference. Any
ideas?
Thanks,
Albert
Albert C. Grobe, PhD
International Heart Institute of Montana Foundation
Tissue Engineering Lab, Saint Patrick Hospital
************************************** See what's free at
http://www.aol.com.
------------------------------
Message: 2
Date: Thu, 5 Jul 2007 14:58:39 -0700 (PDT)
From: Akemi Allison-Tacha <akemiat3377 <@t> yahoo.com>
Subject: Re: [Histonet] Van Gieson question....
To: AGrobe2555 <@t> aol.com, histonet <@t> lists.utsouthwestern.edu
Message-ID: <6799.69564.qm <@t> web31308.mail.mud.yahoo.com>
Content-Type: text/plain; charset=iso-8859-1
The VVG ELASTIC Stain is used for the demonstration of
pathologic changes in elastic fibers. These include
atrophy of the elastic tissue; thinning or loss that
may result from arteriosclerotic changes; and
reduplication, breaks, or splitting that may be used
to demonstrate normal elastic tissue, as in the
identification of veins and arteries, and to determine
whether or not the blood vessels have been invaded by
tumor.
PRINCIPLE OF TEST:
The tissue is over-stained with a soluble lake of
hematoxylin-ferric chloride-iodine. Both ferric
chloride and iodine serve as mordants, but they also
have an oxidizing function that assists in converting
hematoxylin to hematein. The mechanism of dye binding
is probably by formation of hydrogen bonds, but the
chemical groups reacting with the hematoxylin have not
been identified. Because this method requires that
the section be over-stained and then differentiated,
it is a regressive method. Differentiation is
accomplished by using excess mordant, or ferric
chloride, to break the tissue-mordant-dye complex.
The dye will be attracted to the larger amount of
mordant in the differentiating solution and will be
removed from the tissue. The elastic tissue has the
strongest affinity for the iron-hematoxylin complex
and will retain the dye longer than the other tissue
elements. This allows other elements to be
decolorized and the elastic fibers to remain stained.
Sodium thiosulfate is used to remove excess iodine.
van Gieson solution is the most commonly used
counterstain, but others may be used.
COMMENTS AND PRECAUTIONS:
NOTE: It is not necessary to remove mercury deposits
if tissues have been fixed in a mercury-containing
fixative, since they will be removed by the staining
solution.
NOTE: To prepare Working ELASTIC Stain, the reagents
must be added in order given. Prepare in a flask,
swirling as each ingredient is added. This is
critical! DO NOT JUST ADD IN A GRADUATED CYLINDER AND
THEN POUR INTO A COPLIN JAR. This technic will give
inconsistent results!!
Check sections microscopically for adequate
differentiation. Repeat till desired end-point.
Sections may be left a little darker than actually
desired, since they become slightly lighter after the
iodine is removed in sodium thiosulfate. If
differentiation has been carried too far, the sections
may be restained, provided they have not been treated
with alcohol. ***Do not leave in van Gieson Solution
for extended time. ***The picric acid component
decolorizes the elastic fibers.
SPECIMEN REQUIREMENTS:
Specimen consists of any well-fixed tissue. 10%
formalin or Zenker's preferred. Cut section 3-5
microns.
SOLUTIONS:
1. Hematoxylin Solution: "A"
2. Ferric Chloride Solution: "B"
3. Iodine/Iodide Solution: "C"
Working ELASTIC Stain:
Solution "A" 22.0 ml or 30.0 ml
Solution "B" 8.0 ml or 12.0 ml
Solution "C" 8.0 ml or 12.0 ml
4. Differentiation Solution:
5. 5% Sodium Thiosulfate Solution:
6. van Gieson Counterstain:
STORAGE AND STABILITY:
7. Store Solutions in a dark place at room temperature
(18-260C). Hematoxylin Solution: "A", Ferric Chloride
Solution: "B", Iodine/Iodide Solution: "C",
Differentiation Solution are stable for 18 months. 5%
Sodium Thiosulfate Solution and van Gieson
Counterstain are stable for 12 months.
QUALITY ASSURANCE AND CORRECTIVE ACTION GUIDELINES:
Use a section of aorta embedded on edge or a cross
section of a large artery. Check control slide
microscopically after differentiating in ferric
chloride for proper result.
STANDARD STAINING METHOD:
1. Deparaffinize and hydrate to water.
2. Place slides in Working ELASTIC Stain for 15 to 30
minutes. (Working ELASTIC Stain is good for at least
24hrs).
3. Wash sections in running water until no excess
stain remains on slides. Tissue sections should be
intense black and slides will look dirty where they
have been immersed in the staining solution.
4. Dip sections in differentiation solution 15-30
times and transfer to tap water. Check sections
microscopically for adequate differentiation. Repeat
till desired end-point. Sections may be left a little
darker than actually desired, since they become
slightly lighter after the iodine is removed in sodium
thiosulfate. If differentiation has been carried too
far, the sections may be restained, provided they have
not been treated with alcohol.
5. Wash well in running water.
6. Place slides in Sodium thiosulfate Solution for 1
minute.
7. Wash in tap water.
8. Counterstain in van Gieson's Solution 2-5 minutes.
*Longer time as solution ages.
9. Differentiate in 95% alcohol (2 changes), followed
by dehydration in absolute alcohols.
10. Clear in several changes of clearing agent.
11. Mount with resinous mounting media.
RESULTS:
Elastic fibers Blue-black to black
Nuclei Blue to black
Collagen Red
Other tissue elements Yellow
REFERENCES:
Sheehan, D.C. and Hrapchack, B.B., Eds. Theory and
Practice of Histotechnology, 2nd Ed. Mosby, St. Louis,
MO. Pp. 196-197
E. B. Prophet, B. Mills, J.B. Arrington, L.H. Sobin,
A.F.I.P. Laboratory Methods in Histotechnology, 1994,
pp134
Histotechnology A Self-Instructional Text, 2nd Ed. F.
L. Carson, pp.138-139, 1996
Modifications by A. Allison, BIOCARE MEDICAL, Walnut
Creek, CA, 2001
Akemi Allison-Tacha BS, HT (ASCP) HTL
President
Phoenix Lab Consulting & Staffing
Specializing in Histology, SS, IHC, & TMA
Tele: (925) 788-0900
E-Mail: akemiat3377 <@t> yahoo.com
--- AGrobe2555 <@t> aol.com wrote:
> Good afternoon,
> We are doing an Elastic Van Gieson stain on FFPE
> tissues and have come up
> against a bit of a question. While the elastin
> seems to be staining quite
> nicely, the nuclei either are very faintly stained
> or not at all. We have
> shortened the differentiation time in the FeCl, with
> little difference. Any ideas?
> Thanks,
> Albert
>
> Albert C. Grobe, PhD
> International Heart Institute of Montana Foundation
> Tissue Engineering Lab, Saint Patrick Hospital
>
>
>
>
>
> ************************************** See what's
> free at http://www.aol.com.
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
>
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
------------------------------
Message: 3
Date: Fri, 06 Jul 2007 10:50:19 +0300
From: Mikael Niku <mikael.niku <@t> helsinki.fi>
Subject: Re: [Histonet] what do people for ISH
To: "Harvey, Jennifer Lynn" <jennifer.harvey <@t> vanderbilt.edu>
Cc: histonet <@t> lists.utsouthwestern.edu
Message-ID: <468DF43B.60503 <@t> helsinki.fi>
Content-Type: text/plain; charset=ISO-8859-1; format=flowed
Hello Jennifer, Emily, and others,
I think it's really best to be as careful as possible when one is
starting RNA work. But if the lab has been working succesfully for
years, I don't see why they should change the practices. Only if they
are now looking at very rare RNAs, requiring top quality materials for
maximal sensitivity, this might be relevant.
DEPC treatment of solutions is in my experience unnecessary, if one uses
high quality materials. So one can do without this highly toxic stuff.
MilliQ water is as such RNAse free (I tested ours by incubating my RNA
probes a few days at room temp... no detectable degradation). Using
RNAse free glassware & tools, RNAse free chemicals, and of course
gloves, the solutions will be RNAse free without DEPC.
The endogenous RNAses are probably really the most serious threat to
RNA. Tissues like pancreas are a nightmare, due to high enzyme content.
So after killing the animal, you need to work quickly and use effective
RNAse inhibitors / inactivators. We don't have separate processors,
microtomes, or cryotomes for RNA work, and I'm fairly satisfied with the
results.
With best regards, Mikael
------------------------------
Message: 4
Date: Fri, 6 Jul 2007 11:27:02 +0200
From: "Valeria Berno" <valeria.berno <@t> embl.it>
Subject: [Histonet] BUFFER
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<mailman.0.1183741200.8539.histonet <@t> lists.utsouthwestern.edu>
Content-Type: text/plain; charset="windows-1250"
Hi there,
Maybe this is due only to my little experience but I am getting a little bit
confuse on the use of BUFFER!!
Someone use PBS with Ca++ and Mg++, someone else w/o, others KPBS, other PEM
other TBS.......and most of the time the answer of my question is "this is
what we have","It always worked","there is no differences",and so on....
I believe that for instance TBS is used to modify the pH, that PEM and KPBS
eliminates mild aspecific protein binding (because you have more free
charged ions?) and Ca and Mg can bind to cytoskeleton protein.
But I was wondering...what is the rationale in using one compared to another
one.
Thanks in advance for all the comments
GRAZIE
Valeria
Berno Valeria, PhD
EMBL Monterotondo Outstation
via Ramarini 32
00015 Monterotondo Scalo (RM)
Italy
Tel: +39 06 90091 287
Fax: +39 06 90091 272
valeria.berno <@t> embl.it
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------------------------------
Message: 5
Date: Fri, 6 Jul 2007 07:14:56 -0400
From: "Amy Self" <ASelf <@t> gmhsc.com>
Subject: [Histonet] Pathology Cost Analysis
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<39836CD6DB61654E8F95A35898C9218602E4F179 <@t> exchange.gmhpost.com>
Content-Type: text/plain; charset="iso-8859-1"
Good morning Histonetters
Hope everyone had a safe and happy 4th of July...
I need your help once again..... I have to do a cost analysis for
histology and cytology and was wandering if anyone could help me with
this? Thanks in advance.. Amy
Amy Self
Georgetown Hospital Systems
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Message: 6
Date: Fri, 6 Jul 2007 07:34:41 -0500
From: "Jackie M O'Connor" <Jackie.O'Connor <@t> abbott.com>
Subject: [Histonet] Shopping for histology item
To: histonet <@t> lists.utsouthwestern.edu,
histonet-bounces <@t> lists.utsouthwestern.edu
Message-ID:
<OFEB5A14CF.1C4967A6-ON86257310.0044E690-86257310.00452304 <@t> abbott.com>
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I'm trying to find a vendor for foam slide file 'bumpers' or a reasonable
facsimile - the kind that fit in metal slide drawers. We deal with
moving thousands of slides per week, and would spend a whole day cutting
up foam ourselves. They don't have to be foam - just some kind of
stopper to separate groups of slides.
Thanks.
------------------------------
Message: 7
Date: Fri, 6 Jul 2007 08:37:51 -0400
From: "Emily Sours" <talulahgosh <@t> gmail.com>
Subject: Re: [Histonet] what do people for ISH-depc
To: "Mikael Niku" <mikael.niku <@t> helsinki.fi>,
histonet <@t> lists.utsouthwestern.edu
Message-ID:
<b39794b0707060537u49cbee58w97115cddf3ba0308 <@t> mail.gmail.com>
Content-Type: text/plain; charset=ISO-8859-1; format=flowed
The funny thing about DEPC's toxicity is that recently is has been
recently demoted from highly toxic (skull and cross bones symbol) to
irritant (X symbol). Same with powder paraformaldehyde. I wonder how
these decisions are made.
Emily
--
these things happen, you know, you go for a walk in the park one day
and wheelchair ninjas and nazis and pots-and-pans robots show up to
kill you and dinosaurs show up to eat the remains.
------------------------------
Message: 8
Date: Fri, 6 Jul 2007 16:43:43 +0200
From: "Gudrun Lang" <gu.lang <@t> gmx.at>
Subject: AW: [Histonet] Van Gieson question....
To: <AGrobe2555 <@t> aol.com>, <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <000901c7bfdc$0d6a7760$6412a8c0 <@t> dielangs.at>
Content-Type: text/plain; charset="iso-8859-1"
The decreasing of the nuclei stain is due to the acid pH of the vanGieson.
Try to prolong the incubation time in the Weigert's iron hemtaxylin,
afterwards only wash in water, no differentiation and go on with the
protocol to the elastica-dyesolution.
Gudrun Lang
Biomed. Analytikerin
Histolabor
Akh Linz
Krankenhausstr. 9
4020 Linz
+43(0)732/7806-6754
-----Urspr|ngliche Nachricht-----
Von: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] Im Auftrag von
AGrobe2555 <@t> aol.com
Gesendet: Donnerstag, 05. Juli 2007 22:56
An: histonet <@t> lists.utsouthwestern.edu
Betreff: [Histonet] Van Gieson question....
Good afternoon,
We are doing an Elastic Van Gieson stain on FFPE tissues and have come up
against a bit of a question. While the elastin seems to be staining quite
nicely, the nuclei either are very faintly stained or not at all. We have
shortened the differentiation time in the FeCl, with little difference. Any
ideas?
Thanks,
Albert
Albert C. Grobe, PhD
International Heart Institute of Montana Foundation
Tissue Engineering Lab, Saint Patrick Hospital
************************************** See what's free at
http://www.aol.com.
_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
------------------------------
Message: 9
Date: Fri, 6 Jul 2007 12:52:51 -0400
From: "Joyce Cline" <jcline <@t> wchsys.org>
Subject: RE: [Histonet] Shopping for histology item/Jackie
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <000601c7bfee$17a06de0$1d2a14ac <@t> wchsys.org>
Content-Type: text/plain;charset="US-ASCII"
Try Lab Storage Systems, Inc 1-800-345-4167
Foam retainer blocks Catalog # RB-50
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Jackie M
O'Connor
Sent: Friday, July 06, 2007 8:35 AM
To: histonet <@t> lists.utsouthwestern.edu;
histonet-bounces <@t> lists.utsouthwestern.edu
Subject: [Histonet] Shopping for histology item
I'm trying to find a vendor for foam slide file 'bumpers' or a
reasonable
facsimile - the kind that fit in metal slide drawers. We deal with
moving thousands of slides per week, and would spend a whole day cutting
up foam ourselves. They don't have to be foam - just some kind of
stopper to separate groups of slides.
Thanks.
_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
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