[Histonet] IHC H2O2, fatty things, IHC slides
Rene J Buesa
rjbuesa <@t> yahoo.com
Fri Sep 30 08:21:47 CDT 2005
Why I am not surprised about what Ventana did? What took you so long to dump the Ventana instrument?
We always used commercial 3% (Fisher) H202 but never used pure H202 to dilute (too dangerous, too many restrictions, too expensive, too problematic!).
We never added Tween to H202; what we did in order to cut time with our Dako autostainer, was to do the peroxidase block step outside the instrument.
After HIER the slides to PBS/Tween; to a container with 3% H202 for 5 minutes (being totally immesed in 3% H202, Tween was not necessary!); to PBS/Tween to the instrument to start the run with the primary antibody. This way we cut @ 25 min. from each 48 slides run and had no problems at all.
Rene J.
John PJ Coleman <jcolclefa <@t> aol.com> wrote:
H2O2 for IHC :
I tried the store bought and it sucked for us. Ironically we did this
after our peroxide block wasn't working and we discovered that Ventana
had relabelled our expensive H2O2 at their warehouse and sold it to us
6 months after its original expiration date, and under the 2 Ventana
labels was a third label showing that this was drugstore H2O2 marked up
in price a crazy amount. They blamed their drop shipper, then told me
their onsite chemist certified the individual bottles (ok, I don't get
that line, drop shipper + onsite chemist=not logical statement) After
we cancelled that standing order contract and tossed their instrument
in the dumpster behind the hospital, we got concentrated H2O2 from
Sigma and we make our own 3 % in water with no tween. We do notice a
meniscus/surface tension issue on large or fragmented tissue, and we
tried using wash buffer instead of Dh2o, we also added Methanol and
found the methanol breaks the surface tension but evaporates too
quickly. We went back to just dh2o at 300 ul per slide for 10 min and
we don't notice compromised results. We do follow the peroxide with a
casein protein block and our slides are clean. We do up to 500 IHC
slides a day (yes 500 slides a day max. usually 250 - 300 though)
Fatty tissues:
We start with a fixative, 10%NBF with ethanol and glacial acetic acid
added to fix through the fat a little better than NBF alone. Processor
programs have longer 100% ethanols and longer xylene times for better
dehydration and clearing. All solutions at 40C.
IHC slides: understand the tissue adherence will be influenced down the
line by heat retrieval, ph of your retrieval solutions, whether or not
you use enzyme digestion, and if you blue your slides in ammonia water.
We specifically had problems on the slides with the X on em (I don't
have all the brands in my head here at starbucks) when the IHC slides
were precut in the main histo lab on water baths with gelatin or Sta-on
liquid. We have had no issues with the slides with the + or the P, or
the barrier slides from Biogenex. We routinely cut all our slides on a
tap water bath with no additives.
Email me for details if you have questions on any of the above.
On Sep 28, 2005, at 1:02 PM, histonet-request <@t> lists.utsouthwestern.edu
wrote:
Send Histonet mailing list submissions to
histonet <@t> lists.utsouthwestern.edu
To subscribe or unsubscribe via the World Wide Web, visit
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
or, via email, send a message with subject or body 'help' to
histonet-request <@t> lists.utsouthwestern.edu
You can reach the person managing the list at
histonet-owner <@t> lists.utsouthwestern.edu
When replying, please edit your Subject line so it is more specific
than "Re: Contents of Histonet digest..."
Today's Topics:
1. RE: Methyl Green (C.M. van der Loos)
2. Dissolving Alizarin complexon (L.Driessen <@t> orthop.umcn.nl)
3. RE: fatty tissue processing (Rogerson Kemlo (ELHT) Pathology)
4. Superfrost Plus slides (Cathy Malcontenti-Wilson)
5. RE: Hydrogen Peroxide for IHC (Lee & Peggy Wenk)
6. RE: haemoxygenase I or II (Edwards, R.E.)
7. SEM on mouse tissue (Stefano Mantero)
8. RE: Superfrost Plus slides (Smith, Jeffery D. (HSC))
9. RE: Superfrost Plus slides (mucram11 <@t> comcast.net)
10. Source of DIFCO trypsin in the UK
(Stephen.Eyres <@t> sanofi-aventis.com)
11. Re: processing skin and intestines (Gayle Callis)
12. RE: Frozen tissue washing from slide (DeBrosse_Beatrice)
13. Re: Dissolving Alizarin complexon (Gayle Callis)
14. Pap Pen Blues (Andrea T. Hooper)
15. Re: Superfrost Plus slides (Gayle Callis)
16. PAS staining RE: [Histonet] Frozen tissue washing from slide
(Gayle Callis)
17. Re: Pap Pen Blues (Gayle Callis)
18. plastic sections (hstevens <@t> ucsd.edu)
19. Use of methanol and DMSO in Whole mount immunostaining
(Johnson, Teri)
----------------------------------------------------------------------
Message: 1
Date: Wed, 28 Sep 2005 08:55:54 +0200
From: "C.M. van der Loos"
Subject: [Histonet] RE: Methyl Green
To: histonet <@t> lists.utsouthwestern.edu
Cc: MEeva <@t> mednet.ucla.edu
Message-ID: <950f24952f1d.952f1d950f24 <@t> amc.uva.nl>
Content-Type: text/plain; charset="us-ascii"
Hi Mervi,
The brown-red color combination you mentioned allows a
simple
hematoxilin counterstain. I would advise you to work with a
diluted
hematoxilin solution and keep the time short. Check out
the
counterstaining result microscopically and if needed repeat.You
don't
want intensely blue nuclei as this will mask your double staining.
If you really wish to counterstain with methylgreen soak your
slides
in 100 mM Na-acetate buffer pH 5.2 for 15 min. Don't wash.
Cover
sections for 5 min with 0.1% methylgreen (Sigma M6776) dissolved
in
the acetate buffer as above. Wash briefly with with distilled
water
and dry the slides completely at a hot plate. Mount organically
with
VectaMount. This procedure circumvents dehydration by alcohols,
which
isn't good for your red alk. phos. reaction product.
Chris van der Loos, PhD
Dept. of Pathology
Academic Medical Center M2-230
Meibergdreef 9
NL-1105 AZ Amsterdam
The Netherlands
phone: +31 20 5665631
fax: +31 20 6960389
Date: Tue, 27 Sep 2005 14:19:05 -0700
From: "Eeva, Mervi"
Subject: [Histonet] Methyl Green
To: histonet <@t> lists.utsouthwestern.edu
Hi All,
I tried Methyl Green counterstaining on my double stained
immunohistochemistry slides and didn't get any
visible
counterstaining.
Tissue was FFPE brain and otherwise I used mostly Vector reagents
with
DAB
and AP-red detection.
Any ideas what went wrong?
- Mervi Eeva, UCLA
------------------------------
Message: 2
Date: Wed, 28 Sep 2005 09:46:45 +0200
From:
Subject: [Histonet] Dissolving Alizarin complexon
To:
Message-ID: <2E2AC813A84E054C8E8E572131082BE2145510 <@t> umcnet14.umcn.nl>
Content-Type: text/plain; charset="iso-8859-1"
Hello,
Is there anyone who is familiar with dissolving alizarin complexon for
bonelabeling?
We inject this fluorochrome in our testanimals to monitor new
boneformation. Because of that we need to sterilize the solution. For
this we push the solution through a 0,2 um millipore-filter. The
problem is that the filter gets clogged very fast. We've tried
prefiltering through a paperfilter, but that doesn't solve the problem.
Does anyone have a a solution for this?
Greetings,
Léon Driessen,
Orthopaedic Research Lab
UMCN St. Radboud, Nijmegen,
The Netherlands
------------------------------
Message: 3
Date: Wed, 28 Sep 2005 09:05:12 +0100
From: "Rogerson Kemlo \(ELHT\) Pathology"
Subject: RE: [Histonet] fatty tissue processing
To: "Jennifer N. Cresor" ,
Message-ID:
<53A22BBB01D6184EBF7A4DC18A021E9E5AD58B <@t> elht-exch1.xelht.nhs.uk>
Content-Type: text/plain; charset="us-ascii"
This question is a regular; I don't have a protocol as I am a Manager/
Cytologist but from memory fatty tissues are best cut thin, well fixed,
dehydrated and then put into a clearing agent/ degreasant. After the fat
has been 'removed' return to dyhydration, then 'clear' again and
impregnate with wax. You can use many degreasants but I used xylene, Mr.
Lillie 'waxes' eloquently about such matters.
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Jennifer
N. Cresor
Sent: Tuesday, September 27, 2005 5:52 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] fatty tissue processing
Hello,
We are currently in the process of setting up a processor specifically
for
processing fatty tissues. I would really like to get people's protocols
on
processing fatty tissues that have worked well for them. Thank you,
Jennifer
jcresor <@t> icpath.com
_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
------------------------------
Message: 4
Date: Wed, 28 Sep 2005 18:33:11 +1000
From: Cathy Malcontenti-Wilson
Subject: [Histonet] Superfrost Plus slides
To: histonet <@t> lists.utsouthwestern.edu
Message-ID:
<6.2.1.2.2.20050928182518.02658550 <@t> mail.staff.unimelb.edu.au>
Content-Type: text/plain; charset="us-ascii"; format=flowed
Dear All,
OK so question regarding the superfrost slides.
I use them for immunostaining and I remember a few years ago when
cutting
sections and orientating them in the waterbath, if I got it wrong well
bad
luck because the sections were well and truly stuck within a few
seconds.
But now I can orientate them no problem, they lift off easily.
Even though they look like most of the sections are still attached, I am
getting all this strange background immunostaining around the edges of
the
tissues and sometimes it looks as though something has been damaging the
tissue around the edges as well. I remember a while ago there was some
discussion on superfrost slides on histonet but I cant remember what the
conclusion was.
Has anyone noticed any changes with the superfrost slides, or do they
recommend one brand over another?
We use Menzel brand and have tried two suppliers, and both the
superfrost
and the superfrost plus slides.
Thanks in advance.
Cathy
------------------------------
Message: 5
Date: Wed, 28 Sep 2005 04:53:36 -0400
From: "Lee & Peggy Wenk"
Subject: RE: [Histonet] Hydrogen Peroxide for IHC
To:
Message-ID: <1084225373-373221435 <@t> pathology.swmed.edu>
Content-Type: text/plain; charset="us-ascii"
We use "store bought". However, we buy it through our hospital
pharmacy, so
it comes in fresh and cheaper than off the drug store shelf. Plus, we
look
at the expiration date (which is always a year down the road).
Peggy Wenk.HTL(ASCP)SLS
William Beaumont Hospital
Royal Oak, MI 48073
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Rene J
Buesa
Sent: Tuesday, September 27, 2005 5:10 PM
To: Poteete, Jacquie A.; 'CRME (Criss Meligro)';
histonet <@t> pathology.swmed.edu
Subject: RE: [Histonet] Hydrogen Peroxide for IHC
Our lab we always used 3% hydrogen peroxide manufactured by Fisher. We
were
not willing to pay for "brand" 3% H202 like Ventana.
Rene J.
"Poteete, Jacquie A." wrote:
I don't trust drug store Hydrogen Peroxide. You never know how long it
has
been on the shelf or how long it had been sitting in a hot warehouse.
Jacquie Poteete MT(ASCP)QIHC
Lead technologist, IHC Laboratory
Saint Francis Hospital, Tulsa, OK
-----Original Message-----
From: CRME (Criss Meligro) [mailto:meligroc <@t> zgi.com]
Sent: Tuesday, September 27, 2005 2:48 PM
To: histonet <@t> pathology.swmed.edu
Subject: [Histonet] Hydrogen Peroxide for IHC
Just wondering if anyone has purchased 3% Hydrogen Peroxide in the 500
ml
bottle from the local drug store at $1.00 +/- and used it for IHC
instead
of the
$66 bottle from Ventana or similar company? Do you add tween?
thanks....Criss
_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
********* Email Confidentiality Statement *********
Visit http://www.saintfrancis.com/emailconf.asp
_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
---------------------------------
Yahoo! for Good
Click here to donate to the Hurricane Katrina relief effort.
_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
------------------------------
Message: 6
Date: Wed, 28 Sep 2005 10:59:03 +0100
From: "Edwards, R.E."
Subject: [Histonet] RE: haemoxygenase I or II
To:
Message-ID:
Content-Type: text/plain; charset="iso-8859-1"
Wanted antibodies to the above that work on mouse paraffin
processed tissues...thanks
Richard Edwards
MRC TOXICOLOGY UNIT
LEICESTER...U.K.....
------------------------------
Message: 7
Date: Wed, 28 Sep 2005 14:47:23 +0200 (CEST)
From: Stefano Mantero
Subject: [Histonet] SEM on mouse tissue
To: histonet <@t> lists.utsouthwestern.edu
Message-ID: <20050928124723.41442.qmail <@t> web25903.mail.ukl.yahoo.com>
Content-Type: text/plain; charset=iso-8859-1
I need Histo help:
I’m looking for a protocol for a Scanning Electron Microscopy sample
preparation on embryonal mouse tissue.
Please help !
Thanks in advance
Stefano
---------------------------------
Yahoo! Mail: gratis 1GB per i messaggi, antispam, antivirus, POP3
------------------------------
Message: 8
Date: Wed, 28 Sep 2005 09:07:15 -0500
From: "Smith, Jeffery D. \(HSC\)"
Subject: RE: [Histonet] Superfrost Plus slides
To: "Cathy Malcontenti-Wilson"
Cc: histonet <@t> lists.utsouthwestern.edu
Message-ID:
Content-Type: text/plain; charset="iso-8859-1"
We are using VWR Superfrost Plus. We do hundreds of Immunos per week
and have had no such problems.
________________________________
From: histonet-bounces <@t> lists.utsouthwestern.edu on behalf of Cathy
Malcontenti-Wilson
Sent: Wed 9/28/2005 3:33 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Superfrost Plus slides
Dear All,
OK so question regarding the superfrost slides.
I use them for immunostaining and I remember a few years ago when
cutting
sections and orientating them in the waterbath, if I got it wrong well
bad
luck because the sections were well and truly stuck within a few
seconds.
But now I can orientate them no problem, they lift off easily.
Even though they look like most of the sections are still attached, I am
getting all this strange background immunostaining around the edges of
the
tissues and sometimes it looks as though something has been damaging the
tissue around the edges as well. I remember a while ago there was some
discussion on superfrost slides on histonet but I cant remember what the
conclusion was.
Has anyone noticed any changes with the superfrost slides, or do they
recommend one brand over another?
We use Menzel brand and have tried two suppliers, and both the
superfrost
and the superfrost plus slides.
Thanks in advance.
Cathy
_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
------------------------------
Message: 9
Date: Wed, 28 Sep 2005 14:24:09 +0000
From: mucram11 <@t> comcast.net
Subject: RE: [Histonet] Superfrost Plus slides
To: "Smith, Jeffery D. (HSC)" , "Cathy
Malcontenti-Wilson"
Cc: histonet <@t> lists.utsouthwestern.edu
Message-ID:
<092820051424.17323.433AA7890005BBA8000043AB2207001641CECE030E9D0C9A03 <@t> c
omcast.net>
Content-Type: text/plain
Cathy,
You should also be sure you are roatating your slides so the oldest lot
numbers are used first. The coatings are not permenant and can lose
surface attraction over time and with exposure to light, heat and air.
The expiration date is from the time they are manufactured, not when
they arrive to you. Be sure your supplier is sending you fresh slides
that have not been sitting in a warehouse for months with varying
temperatures and conditions.
Hope this helps.
Pam Marcum
-------------- Original message --------------
> We are using VWR Superfrost Plus. We do hundreds of Immunos per week
> and have
> had no such problems.
>
> ________________________________
>
> From: histonet-bounces <@t> lists.utsouthwestern.edu on behalf of Cathy
> Malcontenti-Wilson
> Sent: Wed 9/28/2005 3:33 AM
> To: histonet <@t> lists.utsouthwestern.edu
> Subject: [Histonet] Superfrost Plus slides
>
>
>
> Dear All,
> OK so question regarding the superfrost slides.
> I use them for immunostaining and I remember a few years ago when
> cutting
> sections and orientating them in the waterbath, if I got it wrong well
> bad
> luck because the sections were well and truly stuck within a few
> seconds.
> But now I can orientate them no problem, they lift off easily.
> Even though they look like most of the sections are still attached, I
> am
> getting all this strange background immunostaining around the edges of
> the
> tissues and sometimes it looks as though something has been damaging
> the
> tissue around the edges as well. I remember a while ago there was some
> discussion on superfrost slides on histonet but I cant remember what
> the
> conclusion was.
> Has anyone noticed any changes with the superfrost slides, or do they
> recommend one brand over another?
> We use Menzel brand and have tried two suppliers, and both the
> superfrost
> and the superfrost plus slides.
> Thanks in advance.
> Cathy
>
>
> _______________________________________________
> 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: 10
Date: Wed, 28 Sep 2005 15:41:56 +0100
From: Stephen.Eyres <@t> sanofi-aventis.com
Subject: [Histonet] Source of DIFCO trypsin in the UK
To: histonet <@t> lists.utsouthwestern.edu
Message-ID:
Content-Type: text/plain; charset=ISO-8859-1
Hi,
Could someone give me a source for DIFCO trypsin (0152 or 21530-product
code change???)?
Many thanks
Steve
----------------------------------------------------------
Le présent message ainsi que ses éventuelles pièces jointes est
exclusivement destiné au(x) destinataire(s), personnes physiques ou
morales, qu'il désigne.
Il constitue de ce fait une correspondance à caractère privé et peut
contenir des informations confidentielles.
Si ce message vous est parvenu par erreur, nous vous remercions d'en
aviser
immédiatement l'expéditeur par retour de courrier électronique puis de
le
détruire, ainsi que ses éventuelles pièces jointes, sans en conserver de
copie.
This message, including any attachment, is intended for the use of the
individual or entity to which it is addressed.
It is therefore to be considered as a private correspondence which may
contain confidential information.
If you are not the intended recipient, please advise the sender
immediately
by reply e.mail and delete this message and any attachment thereto
without
retaining a copy.
----------------------------------------------------------
------------------------------
Message: 11
Date: Wed, 28 Sep 2005 09:00:08 -0600
From: Gayle Callis
Subject: Re: [Histonet] processing skin and intestines
To: Christian Franci ,
Histonet <@t> lists.utsouthwestern.edu
Message-ID:
<6.0.0.22.1.20050928085212.01b7f430 <@t> gemini.msu.montana.edu>
Content-Type: text/plain; charset="us-ascii"; format=flowed
Isn't the Citadel a carousel (sp?) style with vacuum on last station of
paraffin? I am not totally familiar with this processor.
With our VIP (vacuum/pressure throughout), 45 minutes per station of
dehyrant, clearing and paraffin (60C) at most but we get superb
=== message truncated ===
---------------------------------
Yahoo! for Good
Click here to donate to the Hurricane Katrina relief effort.
More information about the Histonet
mailing list