[Histonet] Reducing TSA background in ISH

Tue Dec 30 12:21:31 CST 2003

Hello out there,

Does anyone have any experience with using the tyramide signal 
amplification in ISH? I am using it on connective tissue sections with 
Anti-DIG-POD --> DNP Amplification --> Anti-DNP-AP. There is a lot of 
background for all probe concentrations (1:1 through 1:20). I'm 
pretreating the tissues with H2O2. Should I use Levamisol as well? 
Should I block longer (using 1 hour)? Should I block before the 
Anti-DIG and Anti-DNP?

Any suggestions will be very appreciated.

-RK

On Dec 30, 2003, at 1:00 PM, histonet-request <@t> lists.utsouthwestern.edu 
wrote:

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> Today's Topics:
>
>    1. Re: buying books (Lee & Peggy Wenk)
>    2. Minnesota Method for staining Bone Marrows' (mprice26 <@t> juno.com)
>    3. influenza A (Patsy Ruegg)
>    4. CPT Code (Linda McDonald)
>    5. re: IHC background [Peng] - rat/Ms X-Rxn? (David Anthony Wright)
>    6. How to educe back ground (Baowei Peng)
>    7. Jobs in Histology (Tommy Vedilago)
>    8. COBr2 (Molinari, Betsy)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Mon, 29 Dec 2003 13:05:24 -0500
> From: "Lee & Peggy Wenk" <lpwenk <@t> covad.net>
> Subject: Re: [Histonet] buying books
> To: <JCarpenter764 <@t> aol.com>,	<histonet <@t> lists.utsouthwestern.edu>
> Message-ID: <000801c3ce36$5c5165a0$8732fea9 <@t> hppav>
> Content-Type: text/plain;	charset="iso-8859-1"
>
> Order it directly from ASCP. If you are a ASCP member, you receive a 
> 10%
> discount.
>
> https://www.ascp.org/500live/timssnet/products/tnt_products.cfm
>
>
>> From the ASCP webpage:
>
> Histotechnology: A Self-instructional Text 2nd Edition
>
> Price: $95.00
>
> Author: Freida Carson, HT(ASCP), PhD
>
> Advances in histotechnology continue to expand the capabilities of the
> histopathology laboratory, but also require continual study by 
> practitioners
> to remain current. The second edition of Histotechnology; A
> Self-Instructional Text presents all the latest advances as well as the
> fundamentals that are critical to achieving the highest quality work.
> Written as both reference and teaching tool Histotechnology; A
> Self-Instructional Text addresses the essentials of preparing histology
> specimens including staining and processing, laboratory safety issues, 
> and
> immunohistochemistry in fourteen instructional units. Electron 
> microscopy
> and enzyme histochemistry have been added to this edition to help
> histotechnologists preparing for the HTL(ASCP) certifying examination. 
> Each
> unit presents the learning objectives of the section, explains and
> illustrates the technique, and then gives specific learning activities 
> that
> will reinforce the information. Written as both a teaching tool and
> reference, the format also makes troubleshooting fast and easy.
>
> Histotechnology is an excellent learning tool for histologic technic
> students, and for residents and pathologists seeking to expand their
> understanding of the technology used in the histopathology laboratory. 
> It is
> equally well suited as a desk reference for the more common special 
> staining
> techniques and their results.
>
> Partial Contents
> Fixation; Processing; Instrumentation; Safety; Laboratory Mathematics 
> and
> Solution Preparation; Nuclear and Cytoplasmic Staining; Carbohydrates 
> and
> Amyloid; Connective and Muscle Tissue; Nerve; Microorganisms; Pigments,
> Minerals, and Cytoplasmic Granules; Immunohistochemistry; Enzyme
> Histochemistry; Electron Microscopy.
>
> Hardbound; 312 pages; 286 images; 6 tables; 47 figures; 1997
> ISBN: 089189411X
>
> Peggy A. Wenk, HTL(ASCP)SLS
> William Beaumont Hospital
> Royal Oak, MI 48073
>
> ----- Original Message -----
> From: <JCarpenter764 <@t> aol.com>
> To: <histonet <@t> lists.utsouthwestern.edu>
> Sent: Saturday, December 27, 2003 8:19 AM
> Subject: [Histonet] buying books
>
>
>> I recently have tried purchasing histotechnology examinations: the 
>> board
> of
>> registry study guide from amazon....i put my order in november 9th and
> have not
>> received the book. Does anyone know of somewhere else i could try and 
>> find
>> this excellent book.  thanks Jennell
>> _______________________________________________
>> Histonet mailing list
>> Histonet <@t> lists.utsouthwestern.edu
>> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>>
>
>
>
>
> ------------------------------
>
> Message: 2
> Date: Mon, 29 Dec 2003 20:51:01 GMT
> From: mprice26 <@t> juno.com
> Subject: [Histonet] Minnesota Method for staining Bone Marrows'
> To: histonet <@t> lists.utsouthwestern.edu
> Message-ID: <20031229.125149.25056.1398144 <@t> webmail20.nyc.untd.com>
> Content-Type: text/plain
>
>
> Hi Histonetters,
> Hope everyone had a Merry Christmas and will have a Happy New Year.
>
> I need a copy of the Minnesota Method of staining Bone Marrows'. Would 
> anyone happen to have one that they could e-mail to me or fax to me?
>
> Thank you.
>
> Marsha Price
>
> ________________________________________________________________
> The best thing to hit the internet in years - Juno SpeedBand!
> Surf the web up to FIVE TIMES FASTER!
> Only $14.95/ month - visit www.juno.com to sign up today!
>
>
>
> ------------------------------
>
> Message: 3
> Date: Mon, 29 Dec 2003 15:45:30 -0700
> From: "Patsy Ruegg" <pruegg <@t> colobio.com>
> Subject: [Histonet] influenza A
> To: "Histonet <@t> Pathology. Swmed. Edu" <histonet <@t> pathology.swmed.edu>
> Message-ID: <NGBBIMHHKLGNCOCPDKBOKENICDAA.pruegg <@t> colobio.com>
> Content-Type: text/plain;	charset="iso-8859-1"
>
> Looking for a lab that tests for "influenza A" in a paraffin tissue 
> block,
> does that test exist?
> Thanks and Happy New Year!
> Patsy Ruegg
>
>
>
>
> ------------------------------
>
> Message: 4
> Date: Mon, 29 Dec 2003 18:13:30 -0500
> From: "Linda McDonald" <mcdonal1 <@t> ccf.org>
> Subject: [Histonet] CPT Code
> To: histonet <@t> lists.utsouthwestern.edu
> Cc: Gwen Goss <GOSSG.GWCCP1A.GWCCD6 <@t> ccf.org>
> Message-ID: <sff06ed1.047 <@t> cesmtp.ccf.org>
> Content-Type: text/plain; charset=us-ascii
>
> When doing Oil Red O stains, Gomori's Trichrome on Frzen muscle tissue 
> or Pas on frozen muscle tissue, what CPT codes are folks using for 
> this?  Are they counted as an 88313 or would they be an 88314?  Let me 
> know what you all are doing with these.  Thanks a lot and Happy New 
> Year!!!!!
> LGMc
>
>
>
>
>
> ------------------------------
>
> Message: 5
> Date: Mon, 29 Dec 2003 17:37:54 -0600
> From: David Anthony Wright <dw18 <@t> uchicago.edu>
> Subject: [Histonet] re: IHC background [Peng] - rat/Ms X-Rxn?
> To: histonet <@t> lists.utsouthwestern.edu
> Message-ID: <1072741074.3ff0bad2a07e3 <@t> webmail.uchicago.edu>
> Content-Type: text/plain; charset=ISO-8859-1
>
> Hello Baowei Peng (& Histonet)
>
> Amos Brooks has suggested the problem below might be endogenous 
> biotin, with
> helpful suggestions what to do.  I'd like to suggest something else if 
> that
> doesn't work.  A couple of questions-
> 1) Is your anti-mouse secondary a polyclonal serum (almost certainly) 
> and, if
> so, is it not rat-adsorbed?
> 2) Is there any reason for there being an inflammatory reaction in 
> your tissue?
>
> There is about a 5-10% overlap in crossreactivity between anti-mouse 
> and anti-
> rat IgG antibodies (cf. goat & sheep), depending on how distant the 
> host
> species is.  I've used standard Vector biotinylated anti-mouse IgG 
> secondary
> (BA2000) happily for IHC on rat brain for years with no background but 
> got a
> huge background cross reaction in every animal when staining material 
> 2 days
> after brain surgery when inflammation is extensive.  I know it was 
> X-reaction
> with native rat IgGs in the tissue because the problem vanished when I 
> either
> i) added normal rat serum to the 2ndary incubation mix and so competed 
> out the
> cross reacting moiety or ii) used Vector's rat-adsorbed secondary 
> (BA2001).
> It's more expensive of course!
>
> good luck!
> -David Wright
>> Hi,Histoneters
>> I'm doing IHC on rat skeletal muscle. I got a heavy background stain 
>> on
>> all sections and control section without 1st AB. But there is no 
>> stain on
>> the control section without 2nd Ab.
>> It looks like the 2nd Ab will react with the rat tissue. My first Ab 
>> is
>> derived from Mouse, and second Ab is anti-mouse.
>> What could I do to reduce the background stain?-Baowei Peng
> -- 
> David A. Wright, Ph.D.
> University of Chicago
> Section of Neurosurgery
> ================================================
> Does 2+2=5 for large values of 2?  [YES!]
>
>
>
> ------------------------------
>
> Message: 6
> Date: Tue, 30 Dec 2003 9:52:48 +0800
> From: "Baowei Peng" <pengbaowei <@t> 21cn.com>
> Subject: [Histonet] How to educe back ground
> To: Histonet <@t> lists.utsouthwestern.edu
> 	<Histonet <@t> lists.utsouthwestern.edu>
> Message-ID: <2S968769905369.06076 <@t> send6.inner-21cn.com>
> Content-Type: text/plain;      charset="GB2312"
>
> Hi,all
> Thanks for your kindly responses.
> My second Ab is said to be a mAb in the description file from 
> manufacture. But do not say if it was absorbed by rat IgG, problely 
> not.
> David Wright is right. There is an inflammatory reaction as seen by 
> mononuclear cell infiltration and diapedesis in the tissue.
> So maybe add rat serum to blocking and antibody diluent reagent will 
> work. I will try this out.
>
> 				
>
> ¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡Baowei Peng
> ¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡pengbaowei <@t> 21cn.com
> ¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡2003-12-30
>
>
>
>
>
> ------------------------------
>
> Message: 7
> Date: Tue, 30 Dec 2003 06:00:50 -0800
> From: "Tommy Vedilago" <tvedilago <@t> system1.net>
> Subject: [Histonet] Jobs in Histology
> To: "Histonet (E-mail)" <histonet <@t> lists.utsouthwestern.edu>
> Message-ID: <D0019D018F824D45A85B4CC8A9260E9726F1E7 <@t> s1-dc.system1.net>
> Content-Type: text/plain;	charset="iso-8859-1"
>
> Hello again Histonetters,
> We are still looking for highly qualified and energetic managers for 
> several locations across the country. We need supervisors in 
> Washington, D.C., Philadelphia, Maine, Houston, NYC, Tucson, AZ, 
> Tampa, New Jersey, Nashville, and Brooklyn. We are also looking for 
> techs in various locations and we are now accepting travelers who are 
> tired of dealing with the large companies. Please feel free to contact 
> me via email or at 866-797-8361. I hope all of you had a merry 
> Christmas and have a happy New Year.
>
> Tommy Vedilago
> System 1 Search
> (864) 627-0012
> (864) 627-0013 Fax
>
>
>
> ------------------------------
>
> Message: 8
> Date: Tue, 30 Dec 2003 11:41:26 -0600
> From: "Molinari, Betsy" <BMolinari <@t> heart.thi.tmc.edu>
> Subject: [Histonet] COBr2
> To: <histonet <@t> pathology.swmed.edu>
> Message-ID: <C124B59C51D3D447878265D53492834E0EFD75 <@t> thimail.THI2.COM>
> Content-Type: text/plain;	charset="us-ascii"
>
> Hi histonetters,
>  A researcher has given us a protocol he found from quite a while ago
> and it calls for a chemical COBr2. The chemical name is carbonyl
> bromide. We cannot find a supplier. Does anyone know about this
> chemical?
> Thanks,
> Betsy Molinari HT (ASCP)
> Texas Heart Institute
> Houston, TX 77225
> 832-355-6524
> 832-355-6812 (fax)
>
>
>
> ------------------------------
>
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> End of Histonet Digest, Vol 1, Issue 191
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