Re: [Histonet] IHC H2O2, fatty things, IHC slides

From:Rene J Buesa

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  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@lists.utsouthwestern.edu 
wrote:

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Today's Topics:

1. RE: Methyl Green (C.M. van der Loos)
2. Dissolving Alizarin complexon (L.Driessen@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@comcast.net)
10. Source of DIFCO trypsin in the UK
(Stephen.Eyres@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@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@lists.utsouthwestern.edu
Cc: MEeva@mednet.ucla.edu
Message-ID: <950f24952f1d.952f1d950f24@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@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@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@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@lists.utsouthwestern.edu
[mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of Jennifer
N. Cresor
Sent: Tuesday, September 27, 2005 5:52 PM
To: histonet@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@icpath.com


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

Message: 4
Date: Wed, 28 Sep 2005 18:33:11 +1000
From: Cathy Malcontenti-Wilson 
Subject: [Histonet] Superfrost Plus slides
To: histonet@lists.utsouthwestern.edu
Message-ID:
<6.2.1.2.2.20050928182518.02658550@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@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@lists.utsouthwestern.edu
[mailto:histonet-bounces@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@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@zgi.com]
Sent: Tuesday, September 27, 2005 2:48 PM
To: histonet@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

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

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@lists.utsouthwestern.edu
Message-ID: <20050928124723.41442.qmail@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



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

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@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@lists.utsouthwestern.edu on behalf of Cathy 
Malcontenti-Wilson
Sent: Wed 9/28/2005 3:33 AM
To: histonet@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@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet




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

Message: 9
Date: Wed, 28 Sep 2005 14:24:09 +0000
From: mucram11@comcast.net
Subject: RE: [Histonet] Superfrost Plus slides
To: "Smith, Jeffery D. (HSC)" , "Cathy
Malcontenti-Wilson" 
Cc: histonet@lists.utsouthwestern.edu
Message-ID:

<092820051424.17323.433AA7890005BBA8000043AB2207001641CECE030E9D0C9A03@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@lists.utsouthwestern.edu on behalf of Cathy
> Malcontenti-Wilson
> Sent: Wed 9/28/2005 3:33 AM
> To: histonet@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@lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>
> _______________________________________________
> Histonet mailing list
> Histonet@lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet

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

Message: 10
Date: Wed, 28 Sep 2005 15:41:56 +0100
From: Stephen.Eyres@sanofi-aventis.com
Subject: [Histonet] Source of DIFCO trypsin in the UK
To: histonet@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

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

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@lists.utsouthwestern.edu
Message-ID:
<6.0.0.22.1.20050928085212.01b7f430@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 

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