[Histonet] IHC Stainers
Neeraj K. Singh
n.singh <@t> biogenex.com
Mon Jun 1 14:59:53 CDT 2009
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Sent: Monday, June 01, 2009 1:02 PM
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Subject: Histonet Digest, Vol 67, Issue 1
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Today's Topics:
1. RE: muscle striations (Tony Henwood)
2. RE: Peroxidase block again................ (Tony Henwood)
3. problem with attaching MMA section to glass slide
(ooi.ting.huay <@t> nhc.com.sg)
4. RE: MAP-2 antibody (Jim Manavis)
5. RE: Microtome Problems (Deborah Faichney)
6. Re: RE: CD11c for murine tissu (anh2006 <@t> med.cornell.edu)
7. Re: Modified Oserhoff Massons Trichrome
(Simoskevitz <@t> Osteotech.com)
8. Processing times (Simoskevitz <@t> Osteotech.com)
9. RE: RE: CD11c for murine tissu (Patsy Ruegg)
10. IHC Stainers..... (Carol Ruffin)
11. Re: IHC Stainers..... (Rene J Buesa)
12. RE: IHC Stainers..... (Blazek, Linda)
----------------------------------------------------------------------
Message: 1
Date: Mon, 1 Jun 2009 09:10:48 +1000
From: "Tony Henwood" <AnthonyH <@t> chw.edu.au>
Subject: RE: [Histonet] muscle striations
To: "Edwards, R.E." <ree3 <@t> leicester.ac.uk>, "Histonet"
<histonet <@t> lists.utsouthwestern.edu>
Message-ID: <B9EAF61856077F47BF9BE2F89AFC555202FB035B <@t> hedwig.nch.kids>
Content-Type: text/plain; charset="iso-8859-7"
Richard,
Cherukian's Phosphotungstic Acid Haematoxylin works well
Principle:
Cherukian's modification employs an eosin solution that stains the erythrocytes red and differentiates them from the blue fibrin.
Fixation: 10% buffered formalin.
Microtomy: paraffin sections at 5ìm.
Controls:
Use brain sections and section of muscle. A good stain will demonstrate the dendrites as blue where as in a bad stain they appear light grey to salmon in colour. Nuclei, fibrin, platelets and muscle will be blue, red cells and collagen appear red. Muscle striations should be well defined.
Reagents:
1. Stock Eosin: - Warning: Flammable - see MSDS
Eosin Y, water soluble (CI 45380) 0.5g
Distilled Water 10ml
80% Ethanol 190ml
2. Working Eosin Solution:
Stock Eosin 10ml
Before use add 50ul glacial acetic acid.
3. 1% Periodic Acid
4. PTAH solution
Haematoxylin (CI 75290) 0.5g
Phosphotungstic Acid 10g
Distilled water 500ml
Dissolve solid ingredients in separate portions of the water. Use gentle heat for Haematoxylin. Combine solutions when cool. Add 0.088g potassium permanganate to ripen. The stain is ready to use.
Procedure:
1. Dewax and hydrate sections to 80% alcohol.
2. Place slides in working eosin solution for 30 seconds.
3. Wash slides in distilled water for a few seconds.
4. Place slides in 1% periodic acid for 20 minutes.
5. Wash slides in water for 3 minutes.
6. Place slides in PTAH solution for 30-90 minutes in 60oC oven. Check from 30 minutes on.
7. Dehydrate, clear and mount.
Results:
Dendrites, nuclei, fibrin, platelets and muscle - blue
Red blood cells and collagen - red.
Notes:
Reference:
1. , C.J., Histologic. 8(4); 105, (1977).
2. Luna, L., Histologic. 5(2); 66, (1975).
Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC) Laboratory Manager & Senior Scientist
Tel: 612 9845 3306
Fax: 612 9845 3318
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145, AUSTRALIA
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Edwards, R.E.
Sent: Thursday, 28 May 2009 6:29 PM
To: Histonet
Subject: [Histonet] muscle striations
Any favourite methods for the above?? I'm aware of Heidenhain's and Mallory's PTAH.
Many thanks
Richard Edwards
University of Leicester
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Message: 2
Date: Mon, 1 Jun 2009 09:51:33 +1000
From: "Tony Henwood" <AnthonyH <@t> chw.edu.au>
Subject: RE: [Histonet] Peroxidase block again................
To: <njoydobro <@t> aol.com>, <Histonet <@t> lists.utsouthwestern.edu>
Message-ID: <B9EAF61856077F47BF9BE2F89AFC555202FB035E <@t> hedwig.nch.kids>
Content-Type: text/plain; charset="us-ascii"
Yep,
You can use alkaline phosphatase labelling instead of peroxidase
Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC) Laboratory Manager & Senior Scientist
Tel: 612 9845 3306
Fax: 612 9845 3318
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145, AUSTRALIA
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of njoydobro <@t> aol.com
Sent: Friday, 29 May 2009 9:20 PM
To: Histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Peroxidase block again................
good morning,
???? I would like to pose this questions again...Is there anyone out there who has eliminated using Peroxidase Block on either IHC or ICC?
Your feedback is very helpful.
Thanks,
Gene
Cleveland Clinic _______________________________________________
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Message: 3
Date: Mon, 1 Jun 2009 09:23:04 +0800
From: ooi.ting.huay <@t> nhc.com.sg
Subject: [Histonet] problem with attaching MMA section to glass slide
To: Histonet <@t> lists.utsouthwestern.edu
Message-ID:
<OFBAF28C45.4FC11B19-ON482575C8.0006D8B0-482575C8.0007557D <@t> singhealth.com.sg>
Content-Type: text/plain; charset="US-ASCII"
To all histonetters,
I am currently working with the MMA embedded section. I need to attach about 100micro thick sample to glass slide prior to staining. The sample is direct from saw microtome. Secure attached is needed so that the section will not detach from glass slide while staining.
I have tried the Haupts adhesive coated slides and bond-rite slides. It doesnt work very well...
Please advice and share your expertists. Any suggestion and feedback are welcome. Thank you very much!
Regards,
Ooi
_________________________________________________
Confidential information. Unauthorized use or disclosure prohibited.
Refer http://www.singhealth.com.sg/ContactUs/#Disclaimer
------------------------------
Message: 4
Date: Mon, 1 Jun 2009 12:25:50 +0930
From: Jim Manavis <jim.manavis <@t> imvs.sa.gov.au>
Subject: RE: [Histonet] MAP-2 antibody
To: "'Lott, Robert'" <Robert.Lott <@t> trinitymedicalonline.com>, Histonet
<histonet <@t> lists.utsouthwestern.edu>
Message-ID: <003e01c9e264$78bfea20$2c6c140a <@t> 41984n>
Content-Type: text/plain; charset="us-ascii"
Robert
If you are using formalin fixed paraffin embedded human material, Sigma have
a very nice mouse monoclonal that works beautifully. The cat no. is M-4403
(clone HM-2).
Cheers
Jim
Jim Manavis
Laboratory Manager
Hanson Institute
Centre for Neurological Diseases
IMVS, Adelaide, SA, 5000
Australia
Phone: 61-08-8222-3668 / 0401120697
FAX: 61-08-8222 3392
email: jim.manavis <@t> imvs.sa.gov.au
Disclaimer: Not this little black duck!
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Lott, Robert
Sent: Saturday, 30 May 2009 2:05 AM
To: histonet <@t> lists.utsouthwestern.edu
Cc: Christa Hladik
Subject: [Histonet] MAP-2 antibody
Hi Everyone,
One of our pathologists is interested in MAP-2 IHC for the following
application:
Microtubule Associated Protein-2 Is a Sensitive Marker of Primary and
Metastatic Neuroblastoma.
Krishnan C, Heerema-McKenney A, Arber DA. Department of Pathology,
Stanford University, Stanford, CA.
Comment: Microtubule associated protein-2 (MAP-2) is a protein expressed
in high levels in cells derived from the neural crest.
It is a very robust marker of neuronal differentiation and has been used
most often in evaluating neuronal differentiation in tumors
of the central nervous system. MAP-2 showed comparable sensitivity in
staining primary neuroblastomas
(pre- and post-treatment samples) as compared to synaptophysin,
chromogranin, CD56, and beta-catenin.
In contrast to other markers of neuroblastoma, MAP-2 did not show
significant cross reactivity to native bone marrow
precursors, thus eliminating a potential source of confusion.
Does anyone use this antibody for this or like applications....
I have looked and am "fairly" sure that there is not an IVD available,
but would still be interested in knowing which antibody you use.
Thanks,
Robert L. Lott, HTL(ASCP) / Manager, Anatomic Pathology
Trinity Medical Center/ LabFirst/ 800 Montclair Road / Birmingham, AL
35213/ 205-592-5387
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Message: 5
Date: Mon, 1 Jun 2009 10:12:00 +0100
From: Deborah Faichney <d.a.faichney <@t> stir.ac.uk>
Subject: RE: [Histonet] Microtome Problems
To: 'Veterinary Services Laboratory' <vetlab <@t> caribsurf.com>,
"histonet <@t> lists.utsouthwestern.edu"
<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<8ED3F2CA5B78E142B8193376C57330F8E198E18F5C <@t> EXCH2007.ad.stir.ac.uk>
Content-Type: text/plain; charset="us-ascii"
Sharon,
There are little springs located behind the knife holder plate . If one of these is lost then it can cause the twisting that you describe. This happened to me years ago when stripping and cleaning one of our Leica 2035 microtome's. Hope this helps.
Debbie
Debbie Faichney
Histopathology
Institute of Aquaculture
University of Stirling
Stirling
Scotland
UK
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Veterinary Services Laboratory
Sent: 29 May 2009 15:12
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Microtome Problems
Hello All
I'm new to the group and histo. (3 years) so far I'm getting a lot of useful tips from all the messages. Thank you.
I have two microtomes that were working almost perfectly before I had a technician service them. Now, the Leica 2035 Jung biocuts' blade holder is twisting to the side (going up at one end). I have tried tightening, slacking, and cleaning the entire assembly to no avail. Does anyone know of where I might be able to source one or even how to fix the one I have.
Secondly, the Leitz 1512 rotary microtome, is now jumping forward and giving a loud clanking noise. It takes huge chunks out of the tissue.
Any and all help is appreciated,
Thank you
Sharon
Ms. Sharon Drayton
Veterinary Services Laboratory
Ministry of Agriculture & Rural Development
The Pine
St. Michael BB11091
Barbados
Tel: (246) 427-5492 or (246) 427-5073
Fax: (246)426-7517
Email: vetlab <@t> caribsurf.com
Website: www.agriculture.gov.bb
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number SC 011159.
------------------------------
Message: 6
Date: Mon, 1 Jun 2009 13:50:45 +0000
From: anh2006 <@t> med.cornell.edu
Subject: Re: [Histonet] RE: CD11c for murine tissu
To: "Patsy Ruegg" <pruegg <@t> ihctech.net>,
histonet-bounces <@t> lists.utsouthwestern.edu, "'gayle callis'"
<gayle.callis <@t> bresnan.net>, "'Histonet'"
<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<726660407-1243864322-cardhu_decombobulator_blackberry.rim.net-1774812037- <@t> bxe1087.bisx.prod.on.blackberry>
Content-Type: text/plain
Correct me if I am wrong but CD11c is predominantly on dendritic cells and F4/80 is for predominantly macrophage/myeloid populations (therefore a good CD11b substitute). Are you sure you should use F4/80 as a CD11c substitute?
-----Original Message-----
From: Patsy Ruegg <pruegg <@t> ihctech.net>
Date: Sat, 30 May 2009 10:44:14
To: 'gayle callis'<gayle.callis <@t> bresnan.net>; 'Histonet'<histonet <@t> lists.utsouthwestern.edu>
Subject: RE: [Histonet] RE: CD11c for murine tissu
An alternative to cd11c for macrophages is rat anti mouse f4/80 which I use
from Serotec, it works in frozen and ffpe tissue.
Patsy
Patsy Ruegg, HT(ASCP)QIHC
IHCtech
12635 Montview Blvd. Ste.215
Aurora, CO 80045
720-859-4060
fax 720-859-4110
www.ihctech.net
www.ihcrg.org
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of gayle callis
Sent: Saturday, May 30, 2009 8:45 AM
To: 'Histonet'
Subject: [Histonet] RE: CD11c for murine tissu
You wrote: Hi all - I'm looking for a good antibody to use on mouse tissue
(frozen)
to look for CD11c - does anyone have a suggestion for a good antibody
source? Thanks in advance - Melissa
We us CD11c, HL3 clone, Armenian Hamster anti Mouse. Negative control is
Armenian Hamster IgG1 from BD Pharmingen/Invitrogen. Tissues are fresh
frozen sections fixed with our favorite acetone/absolute ethanol fixative,
although you can use 4C acetone for 10 min on overnight air dried sections.
I will be happy to send the other fixative recipe if you want it.
Gayle M. Callis
HTL(ASCP)HT,MT
Bozeman MT
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Message: 7
Date: Mon, 1 Jun 2009 10:34:23 -0400
From: Simoskevitz <@t> Osteotech.com
Subject: [Histonet] Re: Modified Oserhoff Massons Trichrome
To: histonet <@t> lists.utsouthwestern.edu
Message-ID:
<OFDF3BAE20.B1225700-ON852575C8.004FE027-852575C8.004FFA7D <@t> Osteotech.com>
Content-Type: text/plain; charset=US-ASCII
Gayle,
I would love if you could find this protocol. It is definitely worth a
try!
Thanks,
Ricki
*********************************************
Ricki Simoskevitz, Research Associate III
Osteotech Inc.
51 James Way
Eatontown, NJ 07724
Phone: (732) 542-2800 X6328
Fax: (732) 935-1298
**********************************************
------------------------------
Message: 8
Date: Mon, 1 Jun 2009 10:45:42 -0400
From: Simoskevitz <@t> Osteotech.com
Subject: [Histonet] Processing times
To: histonet <@t> lists.utsouthwestern.edu
Message-ID:
<OF0895601F.6BB74B83-ON852575C8.00509D16-852575C8.005103DD <@t> Osteotech.com>
Content-Type: text/plain; charset=US-ASCII
I am looking for a good processing schedule for demineralized rat femurs
and liver samples. We have a Sakura Tissue-Tek VIP processor. Our lab
would like to start doing paraffin sections and I am not sure what
processing schedule works best for these samples.
Thanks for any and all help.
Ricki
*********************************************
Ricki Simoskevitz, Research Associate III
Osteotech Inc.
51 James Way
Eatontown, NJ 07724
Phone: (732) 542-2800 X6328
Fax: (732) 935-1298
**********************************************
------------------------------
Message: 9
Date: Mon, 1 Jun 2009 09:09:30 -0600
From: "Patsy Ruegg" <pruegg <@t> ihctech.net>
Subject: RE: [Histonet] RE: CD11c for murine tissu
To: <anh2006 <@t> med.cornell.edu>,
<histonet-bounces <@t> lists.utsouthwestern.edu>, "'gayle callis'"
<gayle.callis <@t> bresnan.net>, "'Histonet'"
<histonet <@t> lists.utsouthwestern.edu>
Message-ID: <9E06F6F6DD5E48DF8859F7C0C51E387E <@t> Patsyoffice>
Content-Type: text/plain; charset="us-ascii"
I beg your pardon, you are correct, I was thinking of cd11b not cd11c, as
F4/80 is for macs not dendritic cells.
Best regards,
Patsy
Patsy Ruegg, HT(ASCP)QIHC
IHCtech, LLC
Fitzsimmons BioScience Park
12635 Montview Blvd. Suite 215
Aurora, CO 80010
P-720-859-4060
F-720-859-4110
wk email pruegg <@t> ihctech.net
web site www.ihctech.net
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-----Original Message-----
From: anh2006 <@t> med.cornell.edu [mailto:anh2006 <@t> med.cornell.edu]
Sent: Monday, June 01, 2009 7:51 AM
To: Patsy Ruegg; histonet-bounces <@t> lists.utsouthwestern.edu; 'gayle callis';
'Histonet'
Subject: Re: [Histonet] RE: CD11c for murine tissu
Correct me if I am wrong but CD11c is predominantly on dendritic cells and
F4/80 is for predominantly macrophage/myeloid populations (therefore a good
CD11b substitute). Are you sure you should use F4/80 as a CD11c substitute?
-----Original Message-----
From: Patsy Ruegg <pruegg <@t> ihctech.net>
Date: Sat, 30 May 2009 10:44:14
To: 'gayle callis'<gayle.callis <@t> bresnan.net>;
'Histonet'<histonet <@t> lists.utsouthwestern.edu>
Subject: RE: [Histonet] RE: CD11c for murine tissu
An alternative to cd11c for macrophages is rat anti mouse f4/80 which I use
from Serotec, it works in frozen and ffpe tissue.
Patsy
Patsy Ruegg, HT(ASCP)QIHC
IHCtech
12635 Montview Blvd. Ste.215
Aurora, CO 80045
720-859-4060
fax 720-859-4110
www.ihctech.net
www.ihcrg.org
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of gayle callis
Sent: Saturday, May 30, 2009 8:45 AM
To: 'Histonet'
Subject: [Histonet] RE: CD11c for murine tissu
You wrote: Hi all - I'm looking for a good antibody to use on mouse tissue
(frozen)
to look for CD11c - does anyone have a suggestion for a good antibody
source? Thanks in advance - Melissa
We us CD11c, HL3 clone, Armenian Hamster anti Mouse. Negative control is
Armenian Hamster IgG1 from BD Pharmingen/Invitrogen. Tissues are fresh
frozen sections fixed with our favorite acetone/absolute ethanol fixative,
although you can use 4C acetone for 10 min on overnight air dried sections.
I will be happy to send the other fixative recipe if you want it.
Gayle M. Callis
HTL(ASCP)HT,MT
Bozeman MT
_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
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Hi Carol,
BioGenex i6000 is an ideal autostainer for lab having both IHC and SS work load. It does both applications all in one all at once.
Have continous and STAT features for loading fresh slides with those already onboard for staining. 60 slides and 60 reagents capacity. Software completely open with research key to create user own protocol like IHC, SS,IF etc.
Open for primary, detection kit etc.
Thanks
Neeraj
------------------------------
Message: 10
Date: Mon, 01 Jun 2009 09:23:06 -0700
From: "Carol Ruffin" <cruffin <@t> valleycare.com>
Subject: [Histonet] IHC Stainers.....
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <4A239DFA020000F200001CE7 <@t> mail.valleycare.com>
Content-Type: text/plain; charset=US-ASCII
I am working for a small volume lab and we are looking for an IHC stainer. I have not been anywhere to see what is out there and would really like some
neutral opinions, regarding experience or knowledge of products on the market right to get an idea of what direction to go in. We are using a Dako Artisan for both special stains and IHC now but will not be able to get antibodies for this stainer in the future. Ideally, we would like to be able to do both, but would be happy with some solid info on just an IHC stainer. Nothing at all against Dako....great instrument...but my boss would like to have more choices. Thank you,
Carole Ruffin HT (ASCP)
This message and any included attachments are from ValleyCare Health System
and are intended only for the addressee(s). The information contained herein
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Message: 11
Date: Mon, 1 Jun 2009 09:27:01 -0700 (PDT)
From: Rene J Buesa <rjbuesa <@t> yahoo.com>
Subject: Re: [Histonet] IHC Stainers.....
To: histonet <@t> lists.utsouthwestern.edu, Carol Ruffin
<cruffin <@t> valleycare.com>
Message-ID: <44696.1451.qm <@t> web65709.mail.ac4.yahoo.com>
Content-Type: text/plain; charset=iso-8859-1
Try to get in contact with a Leica Microsystems representative to find out about the BondMax. Great instrument.
René J.
--- On Mon, 6/1/09, Carol Ruffin <cruffin <@t> valleycare.com> wrote:
From: Carol Ruffin <cruffin <@t> valleycare.com>
Subject: [Histonet] IHC Stainers.....
To: histonet <@t> lists.utsouthwestern.edu
Date: Monday, June 1, 2009, 12:23 PM
I am working for a small volume lab and we are looking for an IHC stainer. I have not been anywhere to see what is out there and would really like some
neutral opinions, regarding experience or knowledge of products on the market right to get an idea of what direction to go in. We are using a Dako Artisan for both special stains and IHC now but will not be able to get antibodies for this stainer in the future. Ideally, we would like to be able to do both, but would be happy with some solid info on just an IHC stainer. Nothing at all against Dako....great instrument...but my boss would like to have more choices. Thank you,
Carole Ruffin HT (ASCP)
This message and any included attachments are from ValleyCare Health System
and are intended only for the addressee(s). The information contained herein
may include trade secrets or privileged or otherwise confidential information.
Unauthorized review, forwarding, printing, copying, distributing, or using such
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it, please promptly delete this message and notify the sender by e-mail.
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Message: 12
Date: Mon, 1 Jun 2009 12:52:54 -0400
From: "Blazek, Linda" <lblazek <@t> digestivespecialists.com>
Subject: RE: [Histonet] IHC Stainers.....
To: 'Carol Ruffin' <cruffin <@t> valleycare.com>,
"histonet <@t> lists.utsouthwestern.edu"
<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<5A2BD13465E061429D6455C8D6B40E39088A7D3131 <@t> IBMB7Exchange.digestivespecialists.com>
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Carole,
I am using BioCare's Intellipath and like it very much. It is an open stainer so you can use what ever reagents you want to use but BioCare's cost for reagents is excellent. Also, it has a continuous access so adding more slides while a run is in process is not a problem. That's great when you have a pathologist that as soon as you have a run on decides he wants another stain. It also has a stat run capability. The technical and sales support staff is excellent. If you would like any more information you can contact me off line.
Linda
Linda Blazek HT (ASCP)
Manager/Supervisor
GI Pathology of Dayton
7415 Brandt Pike
Huber Heights, OH 45424
Phone: (937) 293-4424 ext 7118
Email: lblazek <@t> digestivespecialists.com
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Carol Ruffin
Sent: Monday, June 01, 2009 12:23 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] IHC Stainers.....
I am working for a small volume lab and we are looking for an IHC stainer. I have not been anywhere to see what is out there and would really like some
neutral opinions, regarding experience or knowledge of products on the market right to get an idea of what direction to go in. We are using a Dako Artisan for both special stains and IHC now but will not be able to get antibodies for this stainer in the future. Ideally, we would like to be able to do both, but would be happy with some solid info on just an IHC stainer. Nothing at all against Dako....great instrument...but my boss would like to have more choices. Thank you,
Carole Ruffin HT (ASCP)
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End of Histonet Digest, Vol 67, Issue 1
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