[Histonet] RE: Dragon and Cerner
Patti McDavid
Pmcdavid <@t> mhg.com
Wed Oct 8 14:27:26 CDT 2014
We went live with Cerner in mid June. Our PA and Patholgists are using Dragon and making their
own templates and shortcuts through Dragon. No problems here.
Patti McDavid
Memorial Hospital Gulfport
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of histonet-request <@t> lists.utsouthwestern.edu
Sent: Wednesday, October 08, 2014 12:02 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: Histonet Digest, Vol 131, Issue 9
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Today's Topics:
1. Kim's question - order documentation (Cheryl)
2. RE: Kim's question - order documentation (Martha Ward-Pathology)
3. RE: [EXTERNAL] RE: [Histonet] Kim's question - order
documentation (Kolman, Kimberly D.)
4. Sakura VIP Tissue Processor (Adesupo, Adesuyi (Banjo))
5. Dragon and Cerner (Mike Pence)
6. Symphony wicking (Amber McKenzie)
7. Re: Dragon and Cerner (Sue)
8. RE: Dragon and Cerner (Weems, Joyce K.)
9. RE: Symphony wicking (Cooper, Brian)
10. Mouse FFPE IHC double stain caspase-3 and Ki67 (Melanie Smith)
11. RE: Mouse FFPE IHC double stain caspase-3 and Ki67 (James Watson)
12. RE: Mouse FFPE IHC double stain caspase-3 and Ki67 (James Watson)
13. RE: Symphony wicking (Gauch, Vicki)
14. Re: Symphony wicking (Sue)
----------------------------------------------------------------------
Message: 1
Date: Tue, 7 Oct 2014 10:13:31 -0700
From: Cheryl <tkngflght <@t> yahoo.com>
Subject: [Histonet] Kim's question - order documentation
To: "histonet <@t> lists.utsouthwestern.edu"
<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<1412702011.76602.YahooMailNeo <@t> web161202.mail.bf1.yahoo.com>
Content-Type: text/plain; charset=us-ascii
Kim-
Your histonet question may not be as complicated as it might seem. Sometimes it's easier to look at these things backwards. What is the desired outcome? If there is an order -- say for a GMS--and it wasn't ordered by one of your pathologists, where did it come from? Can you track back and figure out what doctor ordered it and verify it's a valid request so the testing AND billing is appropriate (not fraudulent).
When the surgeon or clinician collects the sample at the surgery or in their office, sometimes they want something specific -- say 'evaluate for fungus'. They may include this in the surgical notes, the office chart -- other places. His support staff will copy this onto the requisition or somewhere you get the request other than the requisition. If you keep copies of the req and other incoming documentation-- you've satisfied the requirement--you can track the source of the order. If you don't, include it in the gross description or notes that are transcribed onto the report so that you have a durable record that you can find (may take a while if it's the archived chart, but you can find it).
This goes back to the requirement that orders can't just come from anyone or for any wild-hair reason-- and you have to be able to substantiate or prove the valid source of an ordered (and billed) test.
Does that help?
Cheryl
Cheryl Kerry, HT(ASCP)
Full Staff Inc.
Staffing the AP Lab - one Great Tech at a time.
281.852.9457 Office
800.756.3309 Phone and Fax
admin <@t> fullstaff.org
------------------------------
Message: 2
Date: Tue, 7 Oct 2014 19:02:39 +0000
From: Martha Ward-Pathology <mward <@t> wakehealth.edu>
Subject: RE: [Histonet] Kim's question - order documentation
To: Cheryl <tkngflght <@t> yahoo.com>, "histonet <@t> lists.utsouthwestern.edu"
<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<B2CECB1B6665A4479056478F6DE3C4AB19873D76 <@t> exchdb6.medctr.ad.wfubmc.edu>
Content-Type: text/plain; charset="iso-8859-1"
I think this is an interesting question. We frequently get phone calls from clinicians asking for ER, PR, Her2 or sometimes just other IHC stains....just yesterday someone wanted CYK 7 and CYK 20 on a cytology block. We ask that they either call the pathologist who signed out the case and get them to order the stains, or with something like the breast panel, ask that they fax or email us, stating exactly what they want, the patient demographics and surgical number, etc. That way at least we have a paper trail for the files should anyone ask why we did the testing.
?
Martha Ward, MT (ASCP) QIHC
Manager
Molecular Diagnostics Lab
Medical Center Boulevard ?\? Winston-Salem, NC 27157 p 336.716.2109 ?\? f 336.716.5890 ?
mward <@t> wakehealth.edu ?
?
?
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Cheryl
Sent: Tuesday, October 07, 2014 1:14 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Kim's question - order documentation
Kim-
Your histonet question may not be as complicated as it might seem. Sometimes it's easier to look at these things backwards. What is the desired outcome? If there is an order -- say for a GMS--and it wasn't ordered by one of your pathologists, where did it come from? Can you track back and figure out what doctor ordered it and verify it's a valid request so the testing AND billing is appropriate (not fraudulent).
When the surgeon or clinician collects the sample at the surgery or in their office, sometimes they want something specific -- say 'evaluate for fungus'. They may include this in the surgical notes, the office chart -- other places. His support staff will copy this onto the requisition or somewhere you get the request other than the requisition. If you keep copies of the req and other incoming documentation-- you've satisfied the requirement--you can track the source of the order. If you don't, include it in the gross description or notes that are transcribed onto the report so that you have a durable record that you can find (may take a while if it's the archived chart, but you can find it).
This goes back to the requirement that orders can't just come from anyone or for any wild-hair reason-- and you have to be able to substantiate or prove the valid source of an ordered (and billed) test.
Does that help?
Cheryl
Cheryl Kerry, HT(ASCP)
Full Staff Inc.
Staffing the AP Lab - one Great Tech at a time.
281.852.9457 Office
800.756.3309 Phone and Fax
admin <@t> fullstaff.org
_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
------------------------------
Message: 3
Date: Tue, 7 Oct 2014 14:33:11 -0500
From: "Kolman, Kimberly D." <Kim.Kolman <@t> va.gov>
Subject: RE: [EXTERNAL] RE: [Histonet] Kim's question - order
documentation
To: Martha Ward-Pathology <mward <@t> wakehealth.edu>, Cheryl
<tkngflght <@t> yahoo.com>, <histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<9C32F30B6662D74A8419DDDB7E66656A091E8662 <@t> VHAV15MSGA1.v15.med.va.gov>
Content-Type: text/plain; charset="iso-8859-1"
Ok I guess I'm coming from a different world; so many things, like adequacy on an FNA, FS or the like are a given, as are ER, PR, etc on tumors. We have access to patient history here so are able to get a more thorough picture of what a clinician is looking for. Of course these are all addressed in the report.
Clinicians asking for something 'wild-hair' are not the last word; our pathologists have the final say on what testing may or may not be done. If the clinicians' request is not honored, I don't see the reason for noting it.
I think we are well covered with our current practice.
Just have to hope CAP thinks so as well.............. :)
Thanks everyone!
Kim
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Martha Ward-Pathology
Sent: Tuesday, October 07, 2014 2:03 PM
To: Cheryl; histonet <@t> lists.utsouthwestern.edu
Subject: [EXTERNAL] RE: [Histonet] Kim's question - order documentation
I think this is an interesting question. We frequently get phone calls from clinicians asking for ER, PR, Her2 or sometimes just other IHC stains....just yesterday someone wanted CYK 7 and CYK 20 on a cytology block. We ask that they either call the pathologist who signed out the case and get them to order the stains, or with something like the breast panel, ask that they fax or email us, stating exactly what they want, the patient demographics and surgical number, etc. That way at least we have a paper trail for the files should anyone ask why we did the testing.
?
Martha Ward, MT (ASCP) QIHC
Manager
Molecular Diagnostics Lab
Medical Center Boulevard ?\? Winston-Salem, NC 27157 p 336.716.2109 ?\? f 336.716.5890 mward <@t> wakehealth.edu ?
?
?
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Cheryl
Sent: Tuesday, October 07, 2014 1:14 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Kim's question - order documentation
Kim-
Your histonet question may not be as complicated as it might seem. Sometimes it's easier to look at these things backwards. What is the desired outcome? If there is an order -- say for a GMS--and it wasn't ordered by one of your pathologists, where did it come from? Can you track back and figure out what doctor ordered it and verify it's a valid request so the testing AND billing is appropriate (not fraudulent).
When the surgeon or clinician collects the sample at the surgery or in their office, sometimes they want something specific -- say 'evaluate for fungus'. They may include this in the surgical notes, the office chart -- other places. His support staff will copy this onto the requisition or somewhere you get the request other than the requisition. If you keep copies of the req and other incoming documentation-- you've satisfied the requirement--you can track the source of the order. If you don't, include it in the gross description or notes that are transcribed onto the report so that you have a durable record that you can find (may take a while if it's the archived chart, but you can find it).
This goes back to the requirement that orders can't just come from anyone or for any wild-hair reason-- and you have to be able to substantiate or prove the valid source of an ordered (and billed) test.
Does that help?
Cheryl
Cheryl Kerry, HT(ASCP)
Full Staff Inc.
Staffing the AP Lab - one Great Tech at a time.
281.852.9457 Office
800.756.3309 Phone and Fax
admin <@t> fullstaff.org
_______________________________________________
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: 4
Date: Tue, 7 Oct 2014 16:32:11 -0500
From: "Adesupo, Adesuyi (Banjo)" <abadesuyi <@t> nrh-ok.com>
Subject: [Histonet] Sakura VIP Tissue Processor
To: "histonet <@t> lists.utsouthwestern.edu"
<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<04EE4F75BB5FB246ADB68D69B74604439468E27921 <@t> MAIL.nrhnt.nrh-ok.com>
Content-Type: text/plain; charset="iso-8859-1"
Hi,
Please i will appreciate it if you guys can tell me which one of the Sakura VIP Tissue Processors is the best. We are trying to get a new tissue processor and we don't want to make any mistake this time around.
Thanks,
Banjo Adesuyi, BSMT, HT (ASCP) HTL, QIHC, QLS Histology Supervisor, Norman Regional Health System, Norman, OK 73071.
Tel: 405- 307- 1145
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Message: 5
Date: Wed, 8 Oct 2014 14:20:01 +0000
From: Mike Pence <mpence <@t> grhs.net>
Subject: [Histonet] Dragon and Cerner
To: "Histonet <@t> lists.utsouthwestern.edu"
<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<D6DF96C438FB8D42B04B2345A9452CF501633BB64B <@t> MAIL-MB02.grhs.net>
Content-Type: text/plain; charset="us-ascii"
Hey everyone,
Just wondering who out there is successfully using Dragon within the AP application of Cerner?
Does it work? Not work? Are you using templates in Dragon or Cerner?
You can PM me if you like. This is a peer benchmarking I would like to compare what we do now to what other Cerner users are doing. I don't want to ask this question in a Cerner forum where Cerner can control the commentary.
Thanks,
Mike Pence
AP Supervisor
------------------------------
Message: 6
Date: Wed, 8 Oct 2014 14:24:35 +0000
From: Amber McKenzie <amber.mckenzie <@t> gastrodocs.net>
Subject: [Histonet] Symphony wicking
To: "histonet <@t> lists.utsouthwestern.edu"
<histonet <@t> lists.utsouthwestern.edu>
Message-ID: <5A33C952BB67F4468AF1F36D739212BC0112552B23 <@t> JERRY.Gia.com>
Content-Type: text/plain; charset="us-ascii"
For those of you who have a Symphony, how do you wick your slides? Do you cut them and put them in a basket to drain and then transfer to the trays? Do you cut them and lean them up against your water bath? Do you out them in the oven for a few minutes before you load them onto the symphony? Thanks!
------------------------------
Message: 7
Date: Wed, 8 Oct 2014 14:29:05 +0000 (UTC)
From: Sue <suetp918 <@t> comcast.net>
Subject: Re: [Histonet] Dragon and Cerner
To: Mike Pence <mpence <@t> grhs.net>
Cc: "Histonet <@t> lists.utsouthwestern.edu"
<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<2115505733.889404.1412778545299.JavaMail.root <@t> comcast.net>
Content-Type: text/plain; charset=utf-8
We use Dragon with CoPath Plus and works great, you can make macros and I know my PA have created some quick texts as well.?? We actually stared with Kurzweil in the 80's and expanded to Dragon speak.?? the only time we do not use Dragon is with Synoptic, although it works, it is labor intensive.
??
Susan T. Paturzo
TJUH
------------------------------
Message: 8
Date: Wed, 8 Oct 2014 14:36:20 +0000
From: "Weems, Joyce K." <Joyce.Weems <@t> emoryhealthcare.org>
Subject: [Histonet] RE: Dragon and Cerner
To: "'Mike Pence'" <mpence <@t> grhs.net>,
"Histonet <@t> lists.utsouthwestern.edu"
<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<E3A4EBD57A691646BCCED4AA5911A030011C373687 <@t> e14mbx12n.Enterprise.emory.net>
Content-Type: text/plain; charset="us-ascii"
We use it successfully for micro. We found there was too much noise and muffling in the background for gross.
Joyce Weems
Pathology Manager
678-843-7376 Phone
678-843-7831 Fax
joyce.weems <@t> emoryhealthcare.org
www.saintjosephsatlanta.org
5665 Peachtree Dunwoody Road
Atlanta, GA 30342
This e-mail, including any attachments is the property of Saint Joseph's Hospital and is intended for the sole use of the intended recipient(s). It may contain information that is privileged and confidential. Any unauthorized review, use, disclosure, or distribution is prohibited. If you are not the intended recipient, please delete this message, and reply to the sender regarding the error in a separate email.
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Mike Pence
Sent: Wednesday, October 08, 2014 10:20 AM
To: Histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Dragon and Cerner
Hey everyone,
Just wondering who out there is successfully using Dragon within the AP application of Cerner?
Does it work? Not work? Are you using templates in Dragon or Cerner?
You can PM me if you like. This is a peer benchmarking I would like to compare what we do now to what other Cerner users are doing. I don't want to ask this question in a Cerner forum where Cerner can control the commentary.
Thanks,
Mike Pence
AP Supervisor
_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
________________________________
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If you have received this message in error, please contact
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------------------------------
Message: 9
Date: Wed, 8 Oct 2014 14:50:50 +0000
From: "Cooper, Brian" <bcooper <@t> chla.usc.edu>
Subject: RE: [Histonet] Symphony wicking
To: "histonet <@t> lists.utsouthwestern.edu"
<histonet <@t> lists.utsouthwestern.edu>, "amber.mckenzie <@t> gastrodocs.net"
<amber.mckenzie <@t> gastrodocs.net>
Message-ID:
<C12647AD4408834C8AB48FB0389C63E396C0C3FE <@t> CHLAEXMBH01.LA.AD.CHLA.ORG>
Content-Type: text/plain; charset=us-ascii
We put our slides into the Symphony trays, then bake them at 75 degrees for 15 minutes in our incubators. Then we stain with our routine H&E program, which omits baking inside the Symphony. It's faster than having a bottleneck of slides stack up in the Symphony's intellique, awaiting baking.
Brian
Thanks,
Brian Cooper, HT (ASCP)
Histology Supervisor,
Path & Lab Medicine
Children's Hospital, Los Angeles
Sent from my Galaxy S3, so please forgive any weird typos . . .
-----Original Message-----
From: Amber McKenzie [amber.mckenzie <@t> gastrodocs.net]
Received: Wednesday, 08 Oct 2014, 7:27AM
To: histonet <@t> lists.utsouthwestern.edu [histonet <@t> lists.utsouthwestern.edu]
Subject: [Histonet] Symphony wicking
For those of you who have a Symphony, how do you wick your slides? Do you cut them and put them in a basket to drain and then transfer to the trays? Do you cut them and lean them up against your water bath? Do you out them in the oven for a few minutes before you load them onto the symphony? Thanks!
_______________________________________________
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------------------------------
Message: 10
Date: Wed, 8 Oct 2014 11:38:01 -0400
From: Melanie Smith <melsmith <@t> udel.edu>
Subject: [Histonet] Mouse FFPE IHC double stain caspase-3 and Ki67
To: histonet <@t> lists.utsouthwestern.edu
Message-ID:
<CAAO8d+ynTMiPnngc+g1nW9Jx7qXe27QJL=9tgyoWS=JvozX3xA <@t> mail.gmail.com>
Content-Type: text/plain; charset=UTF-8
Hi everyone,
My lab is trying to do caspase-3 and Ki67 double IHC stain on mouse ffpe
knee joint sections, and we're having issues optimizing the caspase-3
staining. We originally tried to do fluorescent labels but had too much
autofluorescence even after trying several methods of knocking it down. We
decided to move on to traditional IHC and purchased a double stain IHC kit
from abcam R&Rt on human/mouse tissue (DAB & AP/Red) (
http://www.abcam.com/doublestain-ihc-kit-ramprt-on-humanmouse-tissue-dab-amp-apred-ab183283.html).
We started by tying to optimize both antigens independently first and had
success with the Ki-67 staining (primary rat anti-ki67, eBioscience) with
the abcam anti-rat secondary HRP polymer and DAB from the kit. However, we
still haven???t had any luck with the caspase-3 (primary rabbit anti-caspase
3, cell signaling & abcam anti-rabbit secondary AP polymer and permanent
red). We also tried the caspase-3 staining in cell culture after treating
cells with an apoptosis inducer, and only saw extremely weak staining. In
the FFPE sections, we have used overnight antigen retrieval in citrate
buffer ph 6, at 60 C. In both the cell and FFPE sections we do an overnight
primary antibody incubation at 4C. We haven???t seen any convincing staining
in our FFPE sections (there is some dark pink staining but only in one or
two bone marrow cells per section and it seems pretty diffuse) and also
have noticed a yellow/brown precipitate forming and only showing up in the
bone marrow, it also shows up in our negative antibody control sections. We
also tried several different chromogen development times ranging from 10
min to an hour, and didn???t really see a difference. All sections are of
course deparaffinized in xylenes and rehydrated in ethanols before
starting. All washes are done with TBS-T and sections are permeabilized in
0.1% Triton X100 and blocked in 1%BSA in TBST before primary incubation.
All sections are blocked with BLOXALL Endogenous Peroxidase and Alkaline
Phosphatase Blocking Solution from Vector after primary incubation, but
before secondary incubation.
If anyone has any insight into caspase-3 antibodies or doing IHC double
staining that would be great!
Many thanks,
Melanie
--
Melanie Smith, MS
Research Technician II
Department of Biomedical Engineering
University of Delaware
melsmith <@t> udel.edu
------------------------------
Message: 11
Date: Wed, 8 Oct 2014 15:52:17 +0000
From: James Watson <JWatson <@t> gnf.org>
Subject: RE: [Histonet] Mouse FFPE IHC double stain caspase-3 and Ki67
To: Melanie Smith <melsmith <@t> udel.edu>,
"histonet <@t> lists.utsouthwestern.edu"
<histonet <@t> lists.utsouthwestern.edu>
Message-ID: <A032B25305EC6949AC9AFF23C9620616CA3F9060 <@t> EX5.lj.gnf.org>
Content-Type: text/plain; charset="utf-8"
Melanie,
We do this stain, we run Ventana stainers (no advertisement for Ventana intended) . We had to do this as a sequential double stain since the Caspase 3 needed polymer detection where the Ki67 only needs ABC detection. Caspase 3 was done first. Some other differences: Caspase 3 needed FC receptor block and permeablization. Ki67 needed longer HIER time than Caspase 3 so we did a second short HIER after the Caspase 3 was labeled with DAB.
Jamie
James Watson HT?? ASCP
GNF?? Genomics Institute of the Novartis Research Foundation
Scientific Technical Leader II, Histology
Tel?????? 858-332-4647
Fax???? 858-812-1915
jwatson <@t> gnf.org
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Melanie Smith
Sent: Wednesday, October 08, 2014 8:38 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Mouse FFPE IHC double stain caspase-3 and Ki67
Hi everyone,
My lab is trying to do caspase-3 and Ki67 double IHC stain on mouse ffpe knee joint sections, and we're having issues optimizing the caspase-3 staining. We originally tried to do fluorescent labels but had too much autofluorescence even after trying several methods of knocking it down. We decided to move on to traditional IHC and purchased a double stain IHC kit from abcam R&Rt on human/mouse tissue (DAB & AP/Red) ( http://www.abcam.com/doublestain-ihc-kit-ramprt-on-humanmouse-tissue-dab-amp-apred-ab183283.html).
We started by tying to optimize both antigens independently first and had success with the Ki-67 staining (primary rat anti-ki67, eBioscience) with the abcam anti-rat secondary HRP polymer and DAB from the kit. However, we still haven???t had any luck with the caspase-3 (primary rabbit anti-caspase 3, cell signaling & abcam anti-rabbit secondary AP polymer and permanent red). We also tried the caspase-3 staining in cell culture after treating cells with an apoptosis inducer, and only saw extremely weak staining. In the FFPE sections, we have used overnight antigen retrieval in citrate buffer ph 6, at 60 C. In both the cell and FFPE sections we do an overnight primary antibody incubation at 4C. We haven???t seen any convincing staining in our FFPE sections (there is some dark pink staining but only in one or two bone marrow cells per section and it seems pretty diffuse) and also have noticed a yellow/brown precipitate forming and only showing up in the bone marrow, it also sho
ws up in our negative antibody control sections. We also tried several different chromogen development times ranging from 10 min to an hour, and didn???t really see a difference. All sections are of course deparaffinized in xylenes and rehydrated in ethanols before starting. All washes are done with TBS-T and sections are permeabilized in 0.1% Triton X100 and blocked in 1%BSA in TBST before primary incubation.
All sections are blocked with BLOXALL Endogenous Peroxidase and Alkaline Phosphatase Blocking Solution from Vector after primary incubation, but before secondary incubation.
If anyone has any insight into caspase-3 antibodies or doing IHC double staining that would be great!
Many thanks,
Melanie
--
Melanie Smith, MS
Research Technician II
Department of Biomedical Engineering
University of Delaware
melsmith <@t> udel.edu
_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
------------------------------
Message: 12
Date: Wed, 8 Oct 2014 15:56:25 +0000
From: James Watson <JWatson <@t> gnf.org>
Subject: RE: [Histonet] Mouse FFPE IHC double stain caspase-3 and Ki67
To: James Watson <JWatson <@t> gnf.org>, Melanie Smith <melsmith <@t> udel.edu>,
"histonet <@t> lists.utsouthwestern.edu"
<histonet <@t> lists.utsouthwestern.edu>
Message-ID: <A032B25305EC6949AC9AFF23C9620616CA3F9071 <@t> EX5.lj.gnf.org>
Content-Type: text/plain; charset="utf-8"
Sorry forgot to add loner HIER damaged the Caspase 3 antigen.
James Watson HT?? ASCP
GNF?? Genomics Institute of the Novartis Research Foundation
Scientific Technical Leader II, Histology
Tel?????? 858-332-4647
Fax???? 858-812-1915
jwatson <@t> gnf.org
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of James Watson
Sent: Wednesday, October 08, 2014 8:52 AM
To: Melanie Smith; histonet <@t> lists.utsouthwestern.edu
Subject: RE: [Histonet] Mouse FFPE IHC double stain caspase-3 and Ki67
Melanie,
We do this stain, we run Ventana stainers (no advertisement for Ventana intended) . We had to do this as a sequential double stain since the Caspase 3 needed polymer detection where the Ki67 only needs ABC detection. Caspase 3 was done first. Some other differences: Caspase 3 needed FC receptor block and permeablization. Ki67 needed longer HIER time than Caspase 3 so we did a second short HIER after the Caspase 3 was labeled with DAB.
Jamie
James Watson HT?? ASCP
GNF?? Genomics Institute of the Novartis Research Foundation Scientific Technical Leader II, Histology Tel?????? 858-332-4647 Fax???? 858-812-1915 jwatson <@t> gnf.org
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Melanie Smith
Sent: Wednesday, October 08, 2014 8:38 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Mouse FFPE IHC double stain caspase-3 and Ki67
Hi everyone,
My lab is trying to do caspase-3 and Ki67 double IHC stain on mouse ffpe knee joint sections, and we're having issues optimizing the caspase-3 staining. We originally tried to do fluorescent labels but had too much autofluorescence even after trying several methods of knocking it down. We decided to move on to traditional IHC and purchased a double stain IHC kit from abcam R&Rt on human/mouse tissue (DAB & AP/Red) ( http://www.abcam.com/doublestain-ihc-kit-ramprt-on-humanmouse-tissue-dab-amp-apred-ab183283.html).
We started by tying to optimize both antigens independently first and had success with the Ki-67 staining (primary rat anti-ki67, eBioscience) with the abcam anti-rat secondary HRP polymer and DAB from the kit. However, we still haven???t had any luck with the caspase-3 (primary rabbit anti-caspase 3, cell signaling & abcam anti-rabbit secondary AP polymer and permanent red). We also tried the caspase-3 staining in cell culture after treating cells with an apoptosis inducer, and only saw extremely weak staining. In the FFPE sections, we have used overnight antigen retrieval in citrate buffer ph 6, at 60 C. In both the cell and FFPE sections we do an overnight primary antibody incubation at 4C. We haven???t seen any convincing staining in our FFPE sections (there is some dark pink staining but only in one or two bone marrow cells per section and it seems pretty diffuse) and also have noticed a yellow/brown precipitate forming and only showing up in the bone marrow, it also sho
ws up in our negative antibody control sections. We also tried several different chromogen development times ranging from 10 min to an hour, and didn???t really see a difference. All sections are of course deparaffinized in xylenes and rehydrated in ethanols before starting. All washes are done with TBS-T and sections are permeabilized in 0.1% Triton X100 and blocked in 1%BSA in TBST before primary incubation.
All sections are blocked with BLOXALL Endogenous Peroxidase and Alkaline Phosphatase Blocking Solution from Vector after primary incubation, but before secondary incubation.
If anyone has any insight into caspase-3 antibodies or doing IHC double staining that would be great!
Many thanks,
Melanie
--
Melanie Smith, MS
Research Technician II
Department of Biomedical Engineering
University of Delaware
melsmith <@t> udel.edu
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Message: 13
Date: Wed, 8 Oct 2014 16:47:54 +0000
From: "Gauch, Vicki" <GauchV <@t> mail.amc.edu>
Subject: [Histonet] RE: Symphony wicking
To: Amber McKenzie <amber.mckenzie <@t> gastrodocs.net>,
"histonet <@t> lists.utsouthwestern.edu"
<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
<BF796EC3274FE947B2FD0C2AD637CBE97AA8EA9E <@t> wexcmbx01v.AMCNT.AMC.EDU>
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We have a combination of things that we do. Most people cut their slides and stand them up on the side of their waterbath until they are dry and then put them in a rack and transfer them to the Symphony slide tray. If there is any remaining water on the slide they wipe that off with a Kim Wipe before putting in the tray.
Vicki Gauch
AMCH
Albany, NY
________________________________________
From: histonet-bounces <@t> lists.utsouthwestern.edu [histonet-bounces <@t> lists.utsouthwestern.edu] on behalf of Amber McKenzie [amber.mckenzie <@t> gastrodocs.net]
Sent: Wednesday, October 08, 2014 10:24 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Symphony wicking
For those of you who have a Symphony, how do you wick your slides? Do you cut them and put them in a basket to drain and then transfer to the trays? Do you cut them and lean them up against your water bath? Do you out them in the oven for a few minutes before you load them onto the symphony? Thanks!
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Message: 14
Date: Wed, 8 Oct 2014 16:49:14 +0000 (UTC)
From: Sue <suetp918 <@t> comcast.net>
Subject: Re: [Histonet] Symphony wicking
To: Amber McKenzie <amber.mckenzie <@t> gastrodocs.net>
Cc: histonet <@t> lists.utsouthwestern.edu
Message-ID: <733085896.908920.1412786954000.JavaMail.root <@t> comcast.net>
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Most of my staff put them against their waterbath or hold them in??a tray for some time.?? For the tech that??place them directly on the trays we usually angel the tray and jut use a kimwipe to remove the water at the bottom of the slide before putting it on the stainer.?? We do not place them in an oven prior to staining mostly due to process and oven space.?? But it really is what ever works for each institution.
??
Sue Paturzo
TJUH
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End of Histonet Digest, Vol 131, Issue 9
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