[Histonet] IHC H2O2, fatty things, IHC slides
John PJ Coleman
jcolclefa <@t> aol.com
Wed Sep 28 17:49:55 CDT 2005
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
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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" <c.m.vanderloos <@t> amc.uva.nl>
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" <MEeva <@t> mednet.ucla.edu>
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: <L.Driessen <@t> orthop.umcn.nl>
Subject: [Histonet] Dissolving Alizarin complexon
To: <histonet <@t> lists.utsouthwestern.edu>
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" <Kemlo.Rogerson <@t> elht.nhs.uk>
Subject: RE: [Histonet] fatty tissue processing
To: "Jennifer N. Cresor" <jcresor <@t> lcpath.com>,
<histonet <@t> lists.utsouthwestern.edu>
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
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------------------------------
Message: 4
Date: Wed, 28 Sep 2005 18:33:11 +1000
From: Cathy Malcontenti-Wilson <cmalc <@t> unimelb.edu.au>
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" <lpwenk <@t> sbcglobal.net>
Subject: RE: [Histonet] Hydrogen Peroxide for IHC
To: <histonet <@t> pathology.swmed.edu>
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." <japoteete <@t> saintfrancis.com> 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
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------------------------------
Message: 6
Date: Wed, 28 Sep 2005 10:59:03 +0100
From: "Edwards, R.E." <ree3 <@t> leicester.ac.uk>
Subject: [Histonet] RE: haemoxygenase I or II
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<DC88BEDFD1FC3F468D0376A7C75465F705C75610 <@t> Saffron.cfs.le.ac.uk>
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 <stema_cba <@t> yahoo.it>
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
---------------------------------
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------------------------------
Message: 8
Date: Wed, 28 Sep 2005 09:07:15 -0500
From: "Smith, Jeffery D. \(HSC\)" <Jeffery-Smith <@t> ouhsc.edu>
Subject: RE: [Histonet] Superfrost Plus slides
To: "Cathy Malcontenti-Wilson" <cmalc <@t> unimelb.edu.au>
Cc: histonet <@t> lists.utsouthwestern.edu
Message-ID:
<EA80A7E0A540C44A8A339A3561E35C60063E2614 <@t> PERCHERON.hsc.net.ou.edu>
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)" <Jeffery-Smith <@t> ouhsc.edu>, "Cathy
Malcontenti-Wilson" <cmalc <@t> unimelb.edu.au>
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:
<OFA02FCAA3.450D8A46-ON8025708A.00508C13 <@t> sanofi-aventis.com>
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 <gcallis <@t> montana.edu>
Subject: Re: [Histonet] processing skin and intestines
To: Christian Franci <cfranci <@t> rigel.com>,
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
exchange of
solvents with VIP processing, and we never add heat to
dehydration/clearing
steps - dries out the tissue even more.
For rat, I would do 1 hour on VIP. If you do not have vacuum and
pressure
throughout for each station, then mouse tissue will survive 1 hour
timing
on a carousel style nicely.
Keep in mind animal are less fatty than human, and can easily be
overdehydrated, dried out.
At 05:38 PM 9/27/2005, you wrote:
> Howdy folks!
> We've been asked by some scientists to process skin, cheek pouches and
> intestines from mice for FF/paraffin embedding for IHC.
> I'm just wondering if any of you happen to have a good protocol for
> doing
> so. I know that for rat tissues one tends to use longer processing
> times..
> We just got a Citadel processing machine and I'd like to set up a
> program
> for this.
> thanks a bunch in advance!
>
> C
Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)
------------------------------
Message: 12
Date: Wed, 28 Sep 2005 08:01:23 -0700
From: "DeBrosse_Beatrice" <DeBrosse_Beatrice <@t> Allergan.com>
Subject: RE: [Histonet] Frozen tissue washing from slide
To: <WWmn916 <@t> aol.com>, <histonet <@t> pathology.swmed.edu>
Message-ID:
<D475C767BF2F6D438FD30E3984FCBB110167B5DE <@t> irmail121.irvine.allergan.com>
Content-Type: text/plain; charset="us-ascii"
How long do you air dry the slides? Have you tried to fix the slides in
acetone? Is the 10% formalin cold when you fix the slides? How thick are
the sections?
I really don't see a problem with the Periodic acid, the Schiffs or the
water rinses, but I believe your acid solution is too concentrated after
your counterstaining. Have you tried 1% acid/alcohol (70% alcohol)?
Sincerely,
Beatrice DeBrosse-Serra
BS, HT(ASCP)
Allergan, Inc.
2525 Dupont Drive RD-2A
Irvine, CA 92612
714-246-5116
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of
WWmn916 <@t> aol.com
Sent: Tuesday, September 27, 2005 6:01 PM
To: histonet <@t> pathology.swmed.edu
Subject: [Histonet] Frozen tissue washing from slide
I'm hoping someone can give me some idea why frozen muscle sections
would
suddenly start washing away from slide while trying to do PAS w/wo
stain?
Other muscle panel stains do okay in staining process.....just the PAS
w/wo is
recently having problems.
-We use superfrost plus slides
-Sections are fixed in 10% formalin prior to stain
-Most water rinses are DI. Only one tap water rinse step right after
amylase digest.
-Does periodic acid have to be room temp prior to staining?
-Could Shiffs reagent be too cold?
-does temperature of water rinses affect adherance of sections to
slides?
-We use a 10% acid water rinse after heme staining. I recall if acid
water
is made incorrectly, tissue sections could wash much like they do if
acid
water in H+E stain is too strong.
Thanks for any thoughts and help.
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------------------------------
Message: 13
Date: Wed, 28 Sep 2005 09:08:59 -0600
From: Gayle Callis <gcallis <@t> montana.edu>
Subject: Re: [Histonet] Dissolving Alizarin complexon
To: <L.Driessen <@t> orthop.umcn.nl>, Histonet <@t> lists.utsouthwestern.edu
Message-ID:
<6.0.0.22.1.20050928090319.01b71a78 <@t> gemini.msu.montana.edu>
Content-Type: text/plain; charset="iso-8859-1"; format=flowed
Have you tried a larger pore size, 0.45 um.or larger. One could always
get
the big particles first, then refilter again through smaller pore size
if
you felt it necessary. I have had to do this with normal serums before
when 0.22um clogs up.
Possibly, you could try centrifuging the stock solution, decant off the
top
what you need and microfilter that portion just to eliminate the biggest
particles before you go to microfilter.
Just suggestions here
At 01:46 AM 9/28/2005, you wrote:
> 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
>
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)
------------------------------
Message: 14
Date: Wed, 28 Sep 2005 11:29:57 -0400
From: "Andrea T. Hooper" <anh2006 <@t> med.cornell.edu>
Subject: [Histonet] Pap Pen Blues
To: histonet <@t> lists.utsouthwestern.edu
Message-ID: <p06200700bf606663379b@[140.251.146.71]>
Content-Type: text/plain; charset=us-ascii; format=flowed
Hello All,
I have used Zymed Mini Pap pens for years now as through trials and
tribulations and talking with coworkers we agreed it was the best out
there. The pen rarely came up or leaked and gave beautiful results.
Even withstood heat retrieval in DAKO Target Retrieval Solution with
no problems. The stuff was indestructible!!
This is until recently ... I have noticed with the past several
orders that the formula has changed. The pap pen "ink" is now a blue
green in color (used to be more generic yellow/beige) and it does not
stay on well at all. I end up going home in a state of frustration
every day as I pap pen hundreds of slides with little success for the
entirety of my experiment. Arggggh!
So I have two questions:
(1) Has anyone else noticed this change in formula over the past year
or so? (It may have been longer as I was working with a stock of pap
pens before that)
(2) Who makes your favorite pap pen?
THANKS!
Andrea
--
------------------------------
Message: 15
Date: Wed, 28 Sep 2005 09:29:10 -0600
From: Gayle Callis <gcallis <@t> montana.edu>
Subject: Re: [Histonet] Superfrost Plus slides
To: Cathy Malcontenti-Wilson<cmalc <@t> unimelb.edu.au>,
Histonet <@t> lists.utsouthwestern.edu
Message-ID:
<6.0.0.22.1.20050928090945.01b54cf0 <@t> gemini.msu.montana.edu>
Content-Type: text/plain; charset="us-ascii"; format=flowed
Cathy,
A couple of things, for reorientation of sections, we use the Erie
Polysine
slides (poly l lysine coating) that lets us manipulate sections on
slide at
waterbath. Plus Charge are silane and once the section is attracted to
slide surface, you are done!
Do you use Tween or a detergent in your buffers/diluents before
chromogen
application and throughout the staining procedure? If not what you may
be
experiencing is hydrophobic bonding interactions between tissue and
reagent
proteins (BSA, serums) and ionic interaction of chromogen to slide
surface,
which the detergent will eliminate giving cleaner results. 0.05% Tween
20
is usually adequate to eliminate this problem.
We have used Erie's SuperFrost Plus Charge, their trademark and also
Polysine for section manipulation problems for years, and love them. We
have used Plus charge slides stored long past expiration date without
any
problems with IHC or other routine staining. What other manufacturers
do
for coating slides may vary from Erie products, one can only try them
out
to see if they work well.
As for tissue section damage, can you describe in further detail
please? I
doubt this is caused by the slide coatings but some other problem.
At 02:33 AM 9/28/2005, you wrote:
> 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
Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)
------------------------------
Message: 16
Date: Wed, 28 Sep 2005 09:46:26 -0600
From: Gayle Callis <gcallis <@t> montana.edu>
Subject: PAS staining RE: [Histonet] Frozen tissue washing from slide
To: Histonet <@t> lists.utsouthwestern.edu
Message-ID:
<6.0.0.22.1.20050928093504.01b8d3b8 <@t> gemini.msu.montana.edu>
Content-Type: text/plain; charset="us-ascii"; format=flowed
With frozen sections, a gentle rinse is advisable. We cut frozen
sections
destined for routine stains and go directly to room temperature NBF -
air
drying is not really necessary for routine stains but is for
immunostaiing. Let your frozen sections fix longer than 10 minutes to
ensure good adherance to slide and fixed proteins.
You can proceed with a PAS stain just as you do a paraffin section, at
room
temperature.
Is the 10% acid glacial acetic or hydrochloric acid? You did not specify
which one. If you do acid/alcohol rinse, 0.5 to 1% HCl in 70% for
regressive or Harris hematoxylin , or if you use acetic acid, 4% in
water
is sufficient with progressive Gill type hematoxylin.
> -----Original Message-----
> From: histonet-bounces <@t> lists.utsouthwestern.edu
> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of
> WWmn916 <@t> aol.com
> Sent: Tuesday, September 27, 2005 6:01 PM
> To: histonet <@t> pathology.swmed.edu
> Subject: [Histonet] Frozen tissue washing from slide
>
> I'm hoping someone can give me some idea why frozen muscle sections
> would
> suddenly start washing away from slide while trying to do PAS w/wo
> stain?
> Other muscle panel stains do okay in staining process.....just the PAS
> w/wo is
> recently having problems.
> -We use superfrost plus slides
> -Sections are fixed in 10% formalin prior to stain
> -Most water rinses are DI. Only one tap water rinse step right after
> amylase digest.
> -Does periodic acid have to be room temp prior to staining?
> -Could Shiffs reagent be too cold?
> -does temperature of water rinses affect adherance of sections to
> slides?
> -We use a 10% acid water rinse after heme staining. I recall if acid
> water
> is made incorrectly, tissue sections could wash much like they do if
> acid
> water in H+E stain is too strong.
>
> Thanks for any thoughts and help.
>
>
> _______________________________________________
> 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
Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)
------------------------------
Message: 17
Date: Wed, 28 Sep 2005 10:04:23 -0600
From: Gayle Callis <gcallis <@t> montana.edu>
Subject: Re: [Histonet] Pap Pen Blues
To: "Andrea T. Hooper" <anh2006 <@t> med.cornell.edu>,
Histonet <@t> lists.utsouthwestern.edu
Message-ID:
<6.0.0.22.1.20050928095641.01b98148 <@t> gemini.msu.montana.edu>
Content-Type: text/plain; charset="us-ascii"; format=flowed
Andrea,
We now use Vectors ImmEdge pens - doesn't come up, and has a hint of
color. Cheaper, two to a set. Can remark a slide when wet, although we
do
blot away moisture, but the pen goo does not lift nor flow across a
section
if it contacts buffer - been there and have experienced the "Pap pen oil
slick syndrome" that ruins IHC when section gets goo-coated.
To use ImmEdge, be sure to shake pens very vigorously to redistribute
the
goo (per their instructions). We vortex them hard to do that job.
Maybe
the pens you have now need a good vortexing - worth a try for pricey
little
pens. Pressing the point to start goo is still something that requires
gentle tough, but I have not had too many problems with that. Flow is
generally excellent, right amount.
At 09:29 AM 9/28/2005, you wrote:
> Hello All,
>
> I have used Zymed Mini Pap pens for years now as through trials and
> tribulations and talking with coworkers we agreed it was the best out
> there. The pen rarely came up or leaked and gave beautiful results.
> Even
> withstood heat retrieval in DAKO Target Retrieval Solution with no
> problems. The stuff was indestructible!!
>
> This is until recently ... I have noticed with the past several orders
> that the formula has changed. The pap pen "ink" is now a blue green in
> color (used to be more generic yellow/beige) and it does not stay on
> well
> at all. I end up going home in a state of frustration every day as I
> pap
> pen hundreds of slides with little success for the entirety of my
> experiment. Arggggh!
>
> So I have two questions:
>
> (1) Has anyone else noticed this change in formula over the past year
> or
> so? (It may have been longer as I was working with a stock of pap pens
> before that)
> (2) Who makes your favorite pap pen?
>
> THANKS!
> Andrea
> --
>
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)
------------------------------
Message: 18
Date: Wed, 28 Sep 2005 09:20:45 -0700 (PDT)
From: hstevens <@t> ucsd.edu
Subject: [Histonet] plastic sections
To: histonet <@t> lists.utsouthwestern.edu
Message-ID:
<32021.67.113.110.37.1127924445.squirrel <@t> acs-webmail.ucsd.edu>
Content-Type: text/plain;charset=iso-8859-1
Hi,
I am looking for a good inexpensive lab that can process mouse femurs
(at
present in alcohol) to obtain plastic embedded midshaft sections (30 um
ish). Does anyone have any recommendations for the San Diego area or
beyond? Thanks
Hazel Stevens
------------------------------
Message: 19
Date: Wed, 28 Sep 2005 11:53:22 -0500
From: "Johnson, Teri" <TJJ <@t> Stowers-Institute.org>
Subject: [Histonet] Use of methanol and DMSO in Whole mount
immunostaining
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<C28BAF593DC3314E9C0F3A50191C2E78016CB709 <@t> EXCHKC03.stowers-
institute.org>
Content-Type: text/plain; charset="us-ascii"
Ref: GENES & DEVELOPMENT 9:2509-2522, Misexpression of Hoxa-13 induces
cartilage homeotic transformation and changes cell adhesiveness in chick
limb buds, Yokouchi, et al
In this study, chick embryos were dissected and fixed in methanol/DMSO
(4:1) at 4 degrees C overnight, followed by peroxidase blocking in 5%
hydrogen peroxide in methanol/DMSO (4:1). I am unfamiliar with using
methanol/DMSO as a tissue fixative for whole mount studies. This can
obviously be quite helpful for finding protein targets that are masked
or made unrecognizable using aldehyde fixatives. What effect would the
DMSO have on the samples prior to immunostaining?
Teri Johnson
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, MO 64133
------------------------------
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End of Histonet Digest, Vol 22, Issue 37
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