[Histonet] Re: Histonet Digest, Vol 97, Issue 20

[Madeleine Huey]

Magan,

Your problem is very simple.  First of all, you do not need Harris
Hematoxylin after IHC, because Harris Hematoxylin is a regressive
hematoxylin.  What you need is a progressive hematoxylin, like Mayer,
Gill (I, II, III) & etc.

Try this simple procedure after your DAB chromogen;
1) Counterstain in Gill I (Sigma) or Mayer (American MasterTech) for
0.5 - 1 min (longer if want darker counterstain, or use Gill II/III.
Personal preferences)
2) Wash off excess Hematoxylin with tap water (no need distilled
water, your experiment is done)
3) Blue the Nuclei with PBS or TBS buffer (common buffers used by IHC)
4) Wash with water
5) Dehydrate & cover slip with permanent mounting

You can write or call me if you still need help or have any questions.

Madeleine Huey BS, HTL (ASCP) QIHC
Supervisor-Pathology
El Camino Hospital
Mountain View, CA
madeleine_h <@t> elcaminohospital.org

On Sat, Dec 17, 2011 at 10:00 AM,
<histonet-request <@t> lists.utsouthwestern.edu> wrote:
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> Today's Topics:
>
>   1. 2% low melting agarose gel (awatanabe <@t> tgen.org)
>   2. Re: 2% low melting agarose gel (Emily Sours)
>   3. AUTO: Ramona Nelson is out of the office. (returning
>      01/03/2012) (Ramona_Nelson <@t> bd.com)
>   4. Formalin cost account (Morken, Timothy)
>   5. Looking for a used Biocare Decloaker (Sowmya Kedarnath)
>   6. Re: Looking for a used Biocare Decloaker (Akemi Allison)
>   7. CD31 for rat tissue (Colleen Forster)
>   8. Re: CD31 for rat tissue (Lucie Guernsey)
>   9. Diane Tokugawa/CA/KAIPERM is out of the office.
>      (Diane.Tokugawa <@t> kp.org)
>  10. paraffin recycler (Gudrun Lang)
>  11. Re: paraffin recycler (Rene J Buesa)
>  12. Re: DAB haematoxylin counterstain;        too purple, overpowering
>      IHC (Maxim Peshkov)
>  13. Re: New Lab (mequita praet)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Fri, 16 Dec 2011 18:12:01 +0000
> From: <awatanabe <@t> tgen.org>
> Subject: [Histonet] 2% low melting agarose gel
> To: <histonet <@t> lists.utsouthwestern.edu>
> Message-ID: <CB10D763.A82E%awatanabe <@t> tgen.org>
> Content-Type: text/plain; charset="us-ascii"
>
> I have had much trouble with the world of agarose and cell lines.  I
> finally gave up on low melt agarose and went with the standard DNA grade
> agarose.  I also only make it 0.8% and once I fixed the processor routine
> and a few minor details I've had much better success.  I would be happy to
> discuss with you in detail.  Just let me know.
>
> Aprill Watanabe, B.S.
> Research Associate II
> Integrated Cancer Genomics Division
> Tissue Microarray Center (TMA)
> Macromolecular Analyte Processing Center (MAPC)
> Translational Genomics Research Institute (TGen)
> 445 North 5th Street
> Phoenix, AZ 85004
> Main: 602-343-8822
> Fax: 602-343-8717
> Cell: 602-481-8654
> email: awatanabe <@t> tgen.org
> website: www.tgen.org
>
>
>
> On 12/16/11 11:05 AM, "histonet-request <@t> lists.utsouthwestern.edu"
> <histonet-request <@t> lists.utsouthwestern.edu> wrote:
>
>>2% low melting agarose gel
>
>
>
>
> ------------------------------
>
> Message: 2
> Date: Fri, 16 Dec 2011 13:15:54 -0500
> From: Emily Sours <talulahgosh <@t> gmail.com>
> Subject: Re: [Histonet] 2% low melting agarose gel
> To: awatanabe <@t> tgen.org, histonet <@t> lists.utsouthwestern.edu
> Message-ID:
>        <CAP=XX1x_ttsaK5+PfZSF=AFQ1R448qjdabkyEn9d5-GxPAddkg <@t> mail.gmail.com>
> Content-Type: text/plain; charset=UTF-8
>
> How is low melting point different than DNA grade? I assume it's cheaper,
> anything else?
> I know I could google it, but it's more fun to ask you guys.
>
> Emily
>
> The whole point of this country is if you want to eat garbage, balloon up
> to 600 pounds and die of a heart attack at 43, you can! You are free to do
> so. To me, thatā€™s beautiful.
> --Ron Swanson
>
>
>
> On Fri, Dec 16, 2011 at 1:12 PM, <awatanabe <@t> tgen.org> wrote:
>
>> I have had much trouble with the world of agarose and cell lines.  I
>> finally gave up on low melt agarose and went with the standard DNA grade
>> agarose.  I also only make it 0.8% and once I fixed the processor routine
>> and a few minor details I've had much better success.  I would be happy to
>> discuss with you in detail.  Just let me know.
>>
>> Aprill Watanabe, B.S.
>> Research Associate II
>> Integrated Cancer Genomics Division
>> Tissue Microarray Center (TMA)
>> Macromolecular Analyte Processing Center (MAPC)
>> Translational Genomics Research Institute (TGen)
>> 445 North 5th Street
>> Phoenix, AZ 85004
>> Main: 602-343-8822
>> Fax: 602-343-8717
>> Cell: 602-481-8654
>> email: awatanabe <@t> tgen.org
>> website: www.tgen.org
>>
>>
>>
>> On 12/16/11 11:05 AM, "histonet-request <@t> lists.utsouthwestern.edu"
>> <histonet-request <@t> lists.utsouthwestern.edu> wrote:
>>
>> >2% low melting agarose gel
>>
>>
>> _______________________________________________
>> Histonet mailing list
>> Histonet <@t> lists.utsouthwestern.edu
>> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>>
>
>
> ------------------------------
>
> Message: 3
> Date: Fri, 16 Dec 2011 16:26:18 -0500
> From: Ramona_Nelson <@t> bd.com
> Subject: [Histonet] AUTO: Ramona Nelson is out of the office.
>        (returning      01/03/2012)
> To: histonet <@t> lists.utsouthwestern.edu
> Message-ID:
>        <OF37352C93.00669260-ON85257968.0075C3F4-85257968.0075C3F4 <@t> bd.com>
> Content-Type: text/plain; charset="US-ASCII"
>
>
>
>
>
>   I am out of the office until 01/03/20
>
>
> I
>
>
>
>
> Note:  This  is  an  automated  response  to  your message &quo   t;Histonet  Digest,  Vol  97,  Issue 19"   PM.
>
> This  is  the  only  notification  you will receive whi   person is away.
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> ------------------------------
>
> Message: 4
> Date: Fri, 16 Dec 2011 14:29:53 -0800
> From: "Morken, Timothy" <Timothy.Morken <@t> ucsfmedctr.org>
> Subject: [Histonet] Formalin cost account
> To: "'Histonet <@t> lists.utsouthwestern.edu'"
>        <Histonet <@t> lists.utsouthwestern.edu>
> Message-ID:
>        <8D7C2D242DBD45498006B21122072BF89F5EE5CE <@t> MCINFRWEM003.ucsfmedicalcenter.org>
>
> Content-Type: text/plain; charset=us-ascii
>
>
> Sara wrote:
>
> "I don't suppose that anyone out in HistoPersonLand has done a cost
> comparison between making your own 10% NBF and purchasing a 50-gallon
> drum of 10% NBF, perhaps?"
>
> I uploaded a spreadsheet for this at my Yahoo Groups page, Histoinfo. Anyone can join the group and download the spreadsheet. Just adjust prices for your situation.
>
> There are some other histo-related downloads there as well for IHC validation.
>
> http://pets.groups.yahoo.com/group/histoinfo/
>
>
> Tim Morken
> Supervisor, Histology, IPOX
> UC San Francisco Medical Center
> Box 1656
> 1600 Divisidero St, B217
> San Francisco, CA 94115
> USA
>
> 415.514.6042 (office)
> 415.885.7409 Fax
> tim.morken <@t> ucsfmedctr.org<mailto:tim.morken <@t> ucsfmedctr.org>
>
>
>
> ------------------------------
>
> Message: 5
> Date: Fri, 16 Dec 2011 16:10:16 -0800
> From: Sowmya Kedarnath <sowmyakedarnath <@t> gmail.com>
> Subject: [Histonet] Looking for a used Biocare Decloaker
> To: histonet <@t> lists.utsouthwestern.edu
> Message-ID:
>        <CAK2PWWgLLWMAg-2qF3pLRzLD-QUpU8WTU-zgH=Nu2=a5zq=kSA <@t> mail.gmail.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> Hello Histonetters!
> Anybody interested in selling a used Biocare Decloaker kindly email me
> with your contact details.Would greatly appreciate the help.
> Best
> Sowmya Kedarnath
>
>
>
> ------------------------------
>
> Message: 6
> Date: Fri, 16 Dec 2011 16:19:59 -0800
> From: Akemi Allison <akemiat3377 <@t> yahoo.com>
> Subject: Re: [Histonet] Looking for a used Biocare Decloaker
> To: Sowmya Kedarnath <sowmyakedarnath <@t> gmail.com>
> Cc: histonet <@t> lists.utsouthwestern.edu
> Message-ID: <3993039B-E202-424D-97E7-1C60B18B8A3B <@t> yahoo.com>
> Content-Type: text/plain;       charset=US-ASCII;       delsp=yes;      format=flowed
>
> Why don't you ask your local Biocare rep if they have any Demo's
> available for sale.  They used to have some available for sale.
>
>
> Akemi Allison BS, HT (ASCP) HTL
> Director
> Phoenix Lab Consulting
> Tele: 408.335.9994
> E-Mail: akemiat3377 <@t> yahoo.com
>
> On Dec 16, 2011, at 4:10 PM, Sowmya Kedarnath wrote:
>
>> Hello Histonetters!
>> Anybody interested in selling a used Biocare Decloaker kindly email me
>> with your contact details.Would greatly appreciate the help.
>> Best
>> Sowmya Kedarnath
>>
>> _______________________________________________
>> Histonet mailing list
>> Histonet <@t> lists.utsouthwestern.edu
>> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>
>
> ------------------------------
>
> Message: 7
> Date: Fri, 16 Dec 2011 19:56:31 -0600
> From: Colleen Forster <cforster <@t> umn.edu>
> Subject: [Histonet] CD31 for rat tissue
> To: Histonet <histonet <@t> lists.utsouthwestern.edu>
> Message-ID: <4EEBF6CF.3020202 <@t> umn.edu>
> Content-Type: text/plain; charset="iso-8859-1"
>
> Hello Histonetters,
>
> I am looking for a CD31 that has been proven to work well in rat tissue
> samples...have any of you out there done this with success? I have good
> human and mouse antibodies not rat!
>
> Thanks in advance!
>
> Colleen Forster
> Bionet Histology Research Laboratory
> U of MN
> 612-626-1930
>
> ------------------------------
>
> Message: 8
> Date: Fri, 16 Dec 2011 18:08:54 -0800
> From: Lucie Guernsey <lucie.s.guernsey <@t> gmail.com>
> Subject: Re: [Histonet] CD31 for rat tissue
> To: Colleen Forster <cforster <@t> umn.edu>
> Cc: Histonet <histonet <@t> lists.utsouthwestern.edu>
> Message-ID:
>        <CANQOiZ3=2_y7fB0DewXUPOXrZsKrdAd52aaQYKD5a_huo8_WYg <@t> mail.gmail.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> Colleen,
>
> Unfortunately, I can't help you out, but I'm also interested in this
> question. If you happen to get responses off-list, can you share which rat
> antibodies are suggested by others? Also, would you mind sharing which
> mouse antibody and dilution you found works well?
>
> Thanks!
> Lucie
>
> Lucie Guernsey
> UC San Diego
> Dept. of Pathology
>
>
> On Fri, Dec 16, 2011 at 5:56 PM, Colleen Forster <cforster <@t> umn.edu> wrote:
>
>> Hello Histonetters,
>>
>> I am looking for a CD31 that has been proven to work well in rat tissue
>> samples...have any of you out there done this with success? I have good
>> human and mouse antibodies not rat!
>>
>> Thanks in advance!
>>
>> Colleen Forster
>> Bionet Histology Research Laboratory
>> U of MN
>> 612-626-1930
>>
>> _______________________________________________
>> Histonet mailing list
>> Histonet <@t> lists.utsouthwestern.edu
>> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>>
>>
>
>
> ------------------------------
>
> Message: 9
> Date: Fri, 16 Dec 2011 21:44:46 -0800
> From: Diane.Tokugawa <@t> kp.org
> Subject: [Histonet] Diane Tokugawa/CA/KAIPERM is out of the office.
> To: histonet <@t> lists.utsouthwestern.edu
> Message-ID:
>        <OF50818DB9.A74320AF-ON88257969.001F90C9-88257969.001F90C9 <@t> kp.org>
> Content-Type: text/plain; charset=US-ASCII
>
>
>
> I will be out of the office starting  12/16/2011 and will not return until
> 12/20/2011.
>
> Note:   For Cytology issues, please call Molly  at 8-421-5487,  Eric at
> 8-421-5405, or Wanda 8-421-5426   For Histology / IHC issues, please call
> Client services 8-421-5408 to reach Maria (IHC), Mario at 8-421-4961, Kiran
> at 8-421-5404,  or Wanda at 8-421-5426.
>
> ------------------------------
>
> Message: 10
> Date: Sat, 17 Dec 2011 14:06:44 +0100
> From: "Gudrun Lang" <gu.lang <@t> gmx.at>
> Subject: [Histonet] paraffin recycler
> To: <histonet <@t> lists.utsouthwestern.edu>
> Message-ID: <18AEAD4EAFD74440816FF353ED18534A <@t> dielangs.at>
> Content-Type: text/plain;       charset="us-ascii"
>
> Hi all!
>
> What do you think about paraffin recycler? Can someone recommend a special
> type or company?
>
> Do the majority use paraffindispensers or just filled containers in the
> heating chamber?
>
>
>
> thank you
>
> Gudrun Lang
>
>
>
> Histolab, Linz, Austria
>
>
>
> ------------------------------
>
> Message: 11
> Date: Sat, 17 Dec 2011 07:17:20 -0800 (PST)
> From: Rene J Buesa <rjbuesa <@t> yahoo.com>
> Subject: Re: [Histonet] paraffin recycler
> To: histonet <@t> lists.utsouthwestern.edu, gu.lang <@t> gmx.at
> Message-ID:
>        <1324135040.31432.YahooMailClassic <@t> web65711.mail.ac4.yahoo.com>
> Content-Type: text/plain; charset=iso-8859-1
>
> I always added melted paraffin from a paraffin dispenser into the hot chamber of the embedding station.
> It never crossed my mind to try to recycle paraffin. I strongly recommend a cost study (cost of such instrument if there exists one) against cost of new paraffin to replace "used"
> (reagents and tissue contaminated) paraffin.
> René J.
>
> --- On Sat, 12/17/11, Gudrun Lang <gu.lang <@t> gmx.at> wrote:
>
>
> From: Gudrun Lang <gu.lang <@t> gmx.at>
> Subject: [Histonet] paraffin recycler
> To: histonet <@t> lists.utsouthwestern.edu
> Date: Saturday, December 17, 2011, 8:06 AM
>
>
> Hi all!
>
> What do you think about paraffin recycler? Can someone recommend a special
> type or company?
>
> Do the majority use paraffindispensers or just filled containers in the
> heating chamber?
>
>
>
> thank you
>
> Gudrun Lang
>
>
>
> Histolab, Linz, Austria
>
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>
> ------------------------------
>
> Message: 12
> Date: Sat, 17 Dec 2011 20:01:15 +0300
> From: Maxim Peshkov <Maxim_71 <@t> mail.ru>
> Subject: Re: [Histonet] DAB haematoxylin counterstain;  too purple,
>        overpowering IHC
> To: "Megan French" <megan.french <@t> mcri.edu.au>
> Cc: histonet <@t> lists.utsouthwestern.edu
> Message-ID: <1353970564.20111217200115 <@t> mail.ru>
> Content-Type: text/plain; charset=windows-1251
>
> Megan,
> Your contersain DAB protocol has two weak point:
> 1- ammonia water. I used instead ammonia wash buffer
> (Tris-EDTA). It is very good and soft for IHC sections.
> 2- your blueing time is very short.
> Please try after 20-30 sec in hematoxyline dip for 1-3
> mins slides into wash buffer, then finish as usual and
> you will see clear and crisp counterstain.
> If hematoxyline will be to much, then you can
> differentiate it in 0.25% HCl 5-10-15 dips, then wash
> buffer.
> Hope this help.
> Maxim Peshkov,
> Russia,
> Taganrog.
>
>> From: "Megan French" <megan.french <@t> mcri.edu.au>
>> Subject: [Histonet] DAB haematoxylin counterstain;   too purple,
>>         overpowering IHC
>> To: <histonet <@t> lists.utsouthwestern.edu>
>> Message-ID:
>> <DE098B84182F9B4F966EE97D926888D9B48C31 <@t> murmx.mcri.edu.au>
>> Content-Type: text/plain;       charset="us-ascii"
>>
>> Hi all,
>> I have been working through an immuno using DAB and counterstaining with
>> harris haematoxylin.
>> Protocol for counterstain:
>> 5 min h20
>> 1 dip haematoxylin
>> 1 min h20
>> 2 dips Blue in ammonia
>> 1 min h20
>> Dehydrate, clear, mount
>> I am finding that my slides are coming out realllly purple and are
>> overpowering my DAB staining so much so that I can't even see it!
>> The haematoxylin is new, it was recently changed. I don't think it has
>> anything to do with the DAB/quenching etc im pretty sure is is haem
>> related.
>> Any suggestions would be appreciated!!
>> Megan French
>> Surgical Research; Murdoch Childrens Research Institute
>> E megan.french <@t> mcri.edu.au
>
>                          mailto:Maxim_71 <@t> mail.ru
>
>
>
>
> ------------------------------
>
> Message: 13
> Date: Sat, 17 Dec 2011 11:28:54 -0500
> From: mequita praet <mdpraet <@t> gmail.com>
> Subject: [Histonet] Re: New Lab
> To: histonet <histonet <@t> lists.utsouthwestern.edu>
> Message-ID:
>        <CAL_-ji_1gVHwGC8jFj77T-mV20QbkHtRK9ZtX44i4xkZhoJ6aw <@t> mail.gmail.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> Hi Phillip,
> I see you have gotton lots of responses. I am also available on a
> part-time consultant basis to help set up histology labs. I would love
> to talk with about this opportunity. I am sure you can continue to
> explore the resourse available here on histonet and work your way
> through this project. However, I do believe you are taking on a
> monmental task, why not let a histotech with experience help you with
> the lab set up? You really will have enough to do to handle office set
> up ( medicare requirements, billing, courier service, etc). The
> headaches will many and long.
>
> Your idea of a lab with wide open spaces and no walls demonstrates
> that you could use more in put from a histotech with lab set up
> experience. After 40 years in the histology lab, I could offer you
> that input.   My consultantion fees are small in comparsion to the
> headaches.
> If you would like to see my resume and discuss this further please
> feel free to email me.
> Mequita Praet, HTL(ASCP)SLS
> mdpraet <@t> gmail.com
>
>
>
> ------------------------------
>
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
> End of Histonet Digest, Vol 97, Issue 20
> ****************************************