[Histonet] Inconsistent Sections with Cryostat

[Perow, Elliott S (PEROWES10)]

Hello All,

I have a Leica Cryocut 1800 with a Reichert-Jung 2020 microtome that has sat 
for 8 years and went through a move from one academic building to another.  I 
am trying to section brook trout brain, but am having difficulty getting 
consistent sections.
I will get a good section and then the next section will be a very very thin 
shaving of usually just the top portion of the OCT block.  I have tried 
adjusting the clearance angle, made sure the blade and specimen is secured, 
replaced the blade with a new one, have tried different sectioning speeds, 
different thicknesses of the section and still continually only get a decent 
section every other turn.  I was wondering if anyone has had any similar 
experiences or if maybe the machine just needs a maintenance check due to it 
being an older machine.  I wasn't sure if the advancement mechanism may be off 
or if it is another issue that doesn't seem apparent to me.  Any help would be 
greatly appreciated.

Thanks,

Elliott Perow
Juniata College
Biology POE


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[Histonet] Trivew

[Gerald Davydov]

Please share protocol for Triview from Zetta Corporation for Venatna.
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[Histonet] Vacuum and pressure in tissue processing

[Teri Johnson]

Dear friends,

I recall hearing at a conference (or maybe it was just a casual conversation by 
an expert during a NSH symposium break) that vacuum and pressure in tissue 
processing really accomplishes very little. I do believe that using heat and 
agitation of the solutions provides more activity kinetically and therefore 
makes processing more efficient.

Can someone affirm or deny the efficacy of vacuum and/or pressure in tissue 
processing, please?

Thank you, as always, for your wisdom.

Teri Johnson
Manager, Histology
Genomics Institute for
Novartis Research
Foundation
San Diego, CA
858-332-4752

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[Histonet] RE: Histotechnologist, (ASCP, QIHC certified) and Pathology assistant position in Seattle area

[Yan Liang]

Needing a  Histotechnologist (ASCP, QIHC certified) or Scientist 
(Histopathology) for a Seattle area.  Five years of experience in routine 
histology including special stains, IHC and pathology knowledge.  Send an 
updated resume to yli...@numirabio.com to be considered.  A full job 
description is on the Numirabio.com 
..
Yan Liang  Ph.D.
Senior Director, Histopathology
Numira Biosciences Inc.
2023 120th Ave NE
Bellevue, WA 98005
Phone: 425-777-4950 ext 201
Cell: 425-830-3090
Fax:  425-777-4949
yli...@numirabio.com
www.numirabio.com
www.altaportal.numirabio.com




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Re: [Histonet] Shipping to Countries Outside of the US

[Jan Shivers]

According to our Receiving department, each country/institution has its own
permit requirements for receiving samples from outside of their own
country.  It is up to the shipper to inquire of the receiver as to what
permits are needed to ship to that country (Germany, in your case).  My
source also said that FedEx would also have good information on this, since
they ship nearly everywhere around the globe.

Jan Shivers


On Wed, Oct 2, 2013 at 11:10 AM, Sheila Miller  wrote:

> Hello All,
>
> A question was posed to me by our Cancer Resource Department...
>
> I am wondering if you/lab has had experience with shipping tissue
> specimens outside the country.  We have a study where we may be doing this
> and they are asking if we have to have a special permit to ship outside our
> country.  I believe it is going to Germany.
>
> Thank you,
> Sheila
>
>
> 
>
> This message and attachment(s), if any, is intended for the sole use of
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> of which it is addressed, and may contain information that is
> privileged,confidential and prohibited
> from disclosure under applicable law. If you are not the addressee, or
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> behalf of the addressee, you are hereby notified that you may not use,
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[Histonet] need lever clamp for blade of leica microtome

[Patricia F Lott]

Does anyone have an extra lever clamp for a Leica microtome?  I'd be happy to 
pay for it, and of course, for shipping!
Thanks,
Patty Lott
UAB CMBD Core Lab
205-934-2007
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[Histonet] RE: Validating new Benchmark instrument

[Eytalis, Robert A]

I am thinking there has to be a way to integrate the Validation into the 
Optimization the vendor does. I think you should be able to make microarray 
blocks/slides of at least 10 positive and more for ERPR per CAP during the 
optimization. Now, the vendor will say they only do Optimization, but there 
this is a way to get  them to do the stains for you at least. You can create 
your own document for the Validation. 
When your manager negotiates the cost of the analyzer, see if the vendor can 
help out with the initial expense with an allowance. It sounds like you might 
be past this point. 
Has anyone else done it this way?

Robert A. Eytalis
Laboratory Manager
robert-eyta...@riversidehealthcare.net
Phone: (815) 935-7256 ext. 5186
(815) 935-7535
Fax (815) 935-7068

Riverside Medical Center
350 N. Wall Street - Kankakee, IL 60901


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  |  
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From: histonet-boun...@lists.utsouthwestern.edu 
[histonet-boun...@lists.utsouthwestern.edu] on behalf of Fawn Bomar 
[fawn.bo...@halifaxregional.com]
Sent: Tuesday, October 01, 2013 12:06 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Validating new Benchmark instrument

Hi everyone,



I know that this subject has come up several times and I am sorry to bother 
everyone again but I was hoping to find some help.  We are upgrading our 
Benchmark Immuno machines to the Benchmark XT immuno machine at the end of 
October.  Does anyone have any suggestions on how to approach the validation 
process?  Do we need to validate every antibody that we use as well as the 
kits?  I know that there is also a gray area surrounding how many slides need 
to be run to consider the antibody validated, but what would be the suggested 
amount of slides to validate a new machine as I know this will be very costly.  
Does anybody have a log/checklist/spreadsheet that they would be willing to 
share to help document this validation of the machine and each of the 
antibodies?



Thank you in advance for your help



Fawn Bomar
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[Histonet] Hi

[Helene Degan]

Hi 
Im looking for an acid phosphatase enzyme procedure that might be out there the 
one we tried in the past was difficult to work up and get good reproducible 
results.
Thanks
 
Helene D
Upstate University Medical 
Syracuse, NY
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Re: [Histonet] Gram stain

[Jennifer MacDonald]

We have had great success with the Twort gram stain.



From:   Mesru T 
To: histonet@lists.utsouthwestern.edu
Date:   10/02/2013 10:13 AM
Subject:[Histonet] Gram stain
Sent by:histonet-boun...@lists.utsouthwestern.edu



Hi All,



Does anybody have a protocol for Gram stain on FFPE mouse intestine
sections. We are looking to distinguish between Klebsiella and Vancomycin
Resistant Enterococcus (VRE). I would like to avoid using Picric acid as
some protocols suggest.

Thanks in advance.
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Re: [Histonet] IHC turnaround time

[Kim Donadio]

In less than 8 hours I can get 96 Ihc stains done with our dako link and that 
is two runs 

Sent from my iPhone

On Oct 1, 2013, at 1:15 PM, "McKenzie, Emily"  wrote:

> Hello all,
> I am currently working on a Six Sigma Green Belt project and am looking for 
> some data. Can any one give me their  general turnaround times for IHC, from 
> time of order to time of delivery?
> Thanks for all your help with this,
> 
> Emily K. McKenzie BS, HT(ASCP)
> 
> Memorial Medical Center│701 North First Street│Springfield, IL 62781
> Ph: 217-788-3991│email: mckenzie.em...@mhsil.com
> 
> 
> 
> 
>  
> This message (including any attachments) contains confidential information 
> intended for a specific individual and purpose, and is protected by law. If 
> you are not the intended recipient, you should delete this message. Any 
> disclosure, copying, or distribution of this message, or the taking of any 
> action based on it, is strictly prohibited.
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Re: [Histonet] RE: MOHS IPs

[Kim Donadio]

What detection kit are you using? 

Sent from my iPhone

On Oct 2, 2013, at 10:47 AM, "Bauer, Karen L."  wrote:

> Thanks for the reply,
> 
> We are pre-fixing in a formalin substitute, but we'll give the acetone a try. 
>  I have diluted the protease, but have not gotten any good results.  We'll 
> keep trying with that as well.
> 
> We've tried multiple incubation times for the cytokeratin... From 5 minutes 
> all the way up to 30 minutes... With no luck.
> 
> We are using an enhanced polymer DAB.
> 
> Thanks for the suggestions!  I greatly appreciate it!  :)
> 
> Karen
> 
> Karen L. Bauer HTL/HT (ASCP) | Histology Supervisor | Pathology | MOHS Lab 
> Supervisor | Dermatology | Phone: 715-838-3205 | bauer.ka...@mayo.edu | Mayo 
> Clinic Health System | 1221 Whipple Street | Eau Claire, WI 54702 | 
> mayoclinichealthsystem.org
> 
> 
> 
> 
> -Original Message-
> From: histonet-boun...@lists.utsouthwestern.edu 
> [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of 
> pru...@ihctech.net
> Sent: Wednesday, October 02, 2013 9:36 AM
> To: Barbara Tibbs; Bauer, Karen L.; 'histonet@lists.utsouthwestern.edu'
> Subject: RE: [Histonet] RE: MOHS IPs
> 
> 
>   I  agree with Barbara try fixing in cold acetone/ethanol mix even =
>   just  for  a  couple  of minutes then go directly to buffer do not dry
>   after=x,  I  would dilute the protease 1:4 with buffer (PBS or TBS
>   what  ever  u  u=) and do it for a short time.  Enzyme digestion on
>   frozen  sections  i=ricky  but  fixing a little and diluting the
>   enzyme  should  help  prevent  ov=  digestion.   How  long  are  u
>   incubating  the  primary?   Are  u usin=n enhanced labeled polymer
>   detection?  HRP/Dab or AEC or Alk.P/fast=d?
> 
> 
> 
> 
> 
> 
> 
> 
>  Original Message 
> 
> Subject: [Histonet] RE: MOHS =s
> 
> From: Barbara Tibbs <[1]barbara.ti...@accuratediagnosticlabs.com>
> 
> D=e: Wed, October 02, 2013 5:54 am
> 
> To: "Bauer, Karen L." <[2]bauer.ka...@mayo.edu>,
> 
> "'histonet@lists.utsouthwest=n.edu'"
> 
> <[3]histonet@lists.utsouthwestern.edu>
> 
> 
> 
> Hello Karen,
>   =A
> 
> Back  in the old days of doing IHC manually on mostly fresh, froze   n  
> tissue  I would immediately place the slide with the frozen section
>   into  a=plin  jar  of  acetone  to  fix it. Keep the coplin jar of
>   acetone  in  the  cr=stat.  Leave  it  in  the acetone for only 10
>   minutes.  Try diluting the Pr=ease 2 - 1:1 to make it gentler so it
>   doesn't  "eat"  the  tissue.  This  te=nique worked well when I was
>   doing ER/PR on frozen breast tumors. Not su= it will work with skin
>   but it's worth a try.
> 
> 
> 
> Barbara S. Tib=
> 
> Histology Supervisor
> 
> Accurate Diagnostic Labs
> 
> South Pl=nfield, NJ
> 
> [4]barbara.ti...@accuratediagnosticlabs.com
> 
> 732-839-3374
> 
> C=l: 610-809-6508
> 
> 
> 
> 
> 
> _=_
> 
> From:  [5]histonet-boun...@lists.utsouthwestern.edu
>   <[6]histonet-bounces@lists.utsouthwestern=du>  on behalf of Bauer,
>   Karen L. <[7]bauer.ka...@mayo.edu>
> 
> Sent: Tuesday, October 01, 20 5:22 PM
> 
> To: '[8]=sto...@lists.utsouthwestern.edu'
> 
> Subject: [Histonet] MOHS IPs
> 
> 
> Hi all,
> 
> 
> 
> We  are  in the process of validating some a=ibodies in our MOHS
>   lab.  The Melan A (Mart 1) antibody is working well, =t it could be
>   darker.  We will be increasing the Ab incubation time to se=f that
>   will help.
> 
> 
> 
> As  for  the Pan Keratin, we cannot get it = work at all. We use
>   Protease  2  on  our Ultra stainer for FFPE tissues in=stology, but
>   this  stain in MOHS is placed on fresh, unfixed tissue... by=anual
>   drop  method.  Any  time we've tried to use an enzyme retrieval, th=
>   tissue looks eaten up... even if we incubate if for a minute.
> 
> 
>   =ASince  the tissue is not fixed, I figured that no retrieval step
>   would  be=eded, but the Pan Keratin refuses to work with or without
>   retrieval.
>   =A
> 
> For  those of you in MOHS labs that are using a manual staining me   thod  
> for  Melan A and Pan Keratin, would you be willing to share your
>   protoc= with us?
> 
> 
> 
> Thanks so much!!
> 
> 
> 
> Karen
> 
> 
> 
> K=en L. Bauer HTL/HT (ASCP) | Histology Supervisor | Pathology |
>   MOHS   Lab   S=ervisor  |  Dermatology  |  Phone:  715-838-3205  |
>   [9]bauer.ka...@mayo.edu<[10]mailto:bauer.ka...@mayo.edu> | Mayo Clinic
>   Health  System  |  122=hipple  Street  |  Eau Claire, WI 54702 |
>   [11]mayoclinichealthsystem.org   rg/>
> 
> 
>   =A
> 
> ___
> 
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> 
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> 
> [14]http://lists.utsouthwestern.edu/mailman/l=tinfo/histonet
> 
> 
> 
> _=
> 
> Histonet mailing list
> 
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> 
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> 
> 
> 
> 
> 
> 
> 
> Refe

[Histonet] Benchmark Ultra troubles

[Gudrun Lang]

Hi Ventana-users,

we have a special technical problem with our Ultra. It seems, that the
liquid in the waste-tube is sometime pressed out of the hole on the platform
like a fountain.

You can imagine the nice surprise, when the oil drops from the Ultra-roof
onto the carousel-top. Everything is oily.

The technical service has'nt found a cause until now. The filter in the tube
has been changed.

Has anyone seen a similar problem? Solved the problem?

 

Gudrun Lang

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[Histonet] Gram stain

[Mesru T]

Hi All,



Does anybody have a protocol for Gram stain on FFPE mouse intestine
sections. We are looking to distinguish between Klebsiella and Vancomycin
Resistant Enterococcus (VRE). I would like to avoid using Picric acid as
some protocols suggest.

Thanks in advance.
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[Histonet] Shipping to Countries Outside of the US

[Sheila Miller]

Hello All,

A question was posed to me by our Cancer Resource Department...

I am wondering if you/lab has had experience with shipping tissue specimens 
outside the country.  We have a study where we may be doing this and they are 
asking if we have to have a special permit to ship outside our country.  I 
believe it is going to Germany.

Thank you,
Sheila




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of which it is addressed, and may contain information that is 
privileged,confidential and prohibited 
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[Histonet] Re: MOHS IPs

[Teri Johnson]

Hi Karen,

It could be that your cytokeratin antibody only recognizes a formalin fixed 
epitope conformation and enzyme digestion has nothing to do with it. Also try 
fixing in formalin and then try the stain again with and without the diluted 
enzyme digestion. Brief antigen retrieval (Citrate buffer pH 6.0, 30 minutes @ 
60 degrees C) may also help. We have had good luck on minimally fixed frozen 
section staining using the lower temp AR with some antibodies.

Intermediate filaments are best preserved with alcohol-based fixatives. You 
could also try using 95% ethanol fixative for 5 minutes prior to staining, 
rinse with buffer and proceed with staining, no digestion.

Teri Johnson
Manager, Histology
Genomics Institute for
Novartis Research
Foundation
San Diego, CA
858-332-4752

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RE: [Histonet] thawing and refreezing muscle?

[Lewis, Sarah]

I have tried this method as well and it works just as described below.

Sarah E. Lewis HT, ASCP
The Research Institute at
Nationwide Children's Hospital
Center for Gene Therapy
Neuromuscular Division
Rm WA3110
(614)722-2204


 

-Original Message-
From: Tony Reilly [mailto:tony_rei...@health.qld.gov.au] 
Sent: Tuesday, October 01, 2013 7:15 PM
To: Histonet; Timothy Morken
Cc: jmitch...@neurology.wisc.edu
Subject: Re: [Histonet] thawing and refreezing muscle?

Hi Tim
 
A guy in our histology group gave a presentation debunking the theory that ice 
crystal artefact is irreversible.  His lab receives frozen specimens from 
thousands of kilometres away from labs with no histology staff.  If there was 
ice crystal artefact he defrosts the specimen at room temperature.  Mop the 
specimen with tissues to remove as much OCT and moisture as possible.  Allow 
the specimen to remain at room temperature on the bench for 20-30 minutes 
depending on the size of the specimen to allow evaporation of more moisture.  
Refreeze the specimen with OCT and cut sections.  The result was absence of ice 
crystal artefact but some separation of muscle bundles which does not interfere 
with staining.  It appears the ice crystals do not damage the fibres just cause 
separation.  So removing the moisture eliminates the problem.  If you have a 
lot of muscle perhaps you could separate a small amount to trial the procedure.
 
regards
Tony
 
 
 

Tony Reilly  B.App.Sc. , M.Sc.
Chief Scientist, Anatomical Pathology
Pathology Queensland-PA Laboratory

Health Services Support Agency | Department of Health
 
Level 1, Building 15,Princess Alexandra Hospital Ipswich Road,WOOLLOONGABBA  
Qld4102
Ph: 07 3176 2412
Mob: 0402 139411
Fax: 07 3176 2930
Email: tony_rei...@health.qld.gov.au
Web:  www.health.qld.gov.au/qhcss/
 
 


>>> "Morken, Timothy"  10/2/2013 2:07 am 
>>> >>>
Histonetters, Does anyone have a good method for thawing muscle and refreezing 
for histochemistry? With good results? We have some bulk-frozen muscle 
(centimeter thick) and that is what they want to try.

Tim Morken
Supervisor, Electron Microscopy/Neuromuscular Special Studies Department of 
Pathology UC San Francisco Medical Center

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RE: [Histonet] RE: MOHS IPs

[Bauer, Karen L.]

Thanks for the reply,

We are pre-fixing in a formalin substitute, but we'll give the acetone a try.  
I have diluted the protease, but have not gotten any good results.  We'll keep 
trying with that as well.

We've tried multiple incubation times for the cytokeratin... From 5 minutes all 
the way up to 30 minutes... With no luck.

We are using an enhanced polymer DAB.

Thanks for the suggestions!  I greatly appreciate it!  :)

Karen

Karen L. Bauer HTL/HT (ASCP) | Histology Supervisor | Pathology | MOHS Lab 
Supervisor | Dermatology | Phone: 715-838-3205 | bauer.ka...@mayo.edu | Mayo 
Clinic Health System | 1221 Whipple Street | Eau Claire, WI 54702 | 
mayoclinichealthsystem.org

 


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of 
pru...@ihctech.net
Sent: Wednesday, October 02, 2013 9:36 AM
To: Barbara Tibbs; Bauer, Karen L.; 'histonet@lists.utsouthwestern.edu'
Subject: RE: [Histonet] RE: MOHS IPs


   I  agree with Barbara try fixing in cold acetone/ethanol mix even =
   just  for  a  couple  of minutes then go directly to buffer do not dry
   after=x,  I  would dilute the protease 1:4 with buffer (PBS or TBS
   what  ever  u  u=) and do it for a short time.  Enzyme digestion on
   frozen  sections  i=ricky  but  fixing a little and diluting the
   enzyme  should  help  prevent  ov=  digestion.   How  long  are  u
   incubating  the  primary?   Are  u usin=n enhanced labeled polymer
   detection?  HRP/Dab or AEC or Alk.P/fast=d?

   


 


   
 Original Message 
   
Subject: [Histonet] RE: MOHS =s
   
From: Barbara Tibbs <[1]barbara.ti...@accuratediagnosticlabs.com>
   
D=e: Wed, October 02, 2013 5:54 am
   
To: "Bauer, Karen L." <[2]bauer.ka...@mayo.edu>,
   
"'histonet@lists.utsouthwest=n.edu'"
   
<[3]histonet@lists.utsouthwestern.edu>
   

   
Hello Karen,
   =A
   
Back  in the old days of doing IHC manually on mostly fresh, froze   n  tissue  
I would immediately place the slide with the frozen section
   into  a=plin  jar  of  acetone  to  fix it. Keep the coplin jar of
   acetone  in  the  cr=stat.  Leave  it  in  the acetone for only 10
   minutes.  Try diluting the Pr=ease 2 - 1:1 to make it gentler so it
   doesn't  "eat"  the  tissue.  This  te=nique worked well when I was
   doing ER/PR on frozen breast tumors. Not su= it will work with skin
   but it's worth a try.
   

   
Barbara S. Tib=
   
Histology Supervisor
   
Accurate Diagnostic Labs
   
South Pl=nfield, NJ
   
[4]barbara.ti...@accuratediagnosticlabs.com
   
732-839-3374
   
C=l: 610-809-6508
   

   

   
_=_
   
From:  [5]histonet-boun...@lists.utsouthwestern.edu
   <[6]histonet-bounces@lists.utsouthwestern=du>  on behalf of Bauer,
   Karen L. <[7]bauer.ka...@mayo.edu>
   
Sent: Tuesday, October 01, 20 5:22 PM
   
To: '[8]=sto...@lists.utsouthwestern.edu'
   
Subject: [Histonet] MOHS IPs

   
Hi all,
   

   
We  are  in the process of validating some a=ibodies in our MOHS
   lab.  The Melan A (Mart 1) antibody is working well, =t it could be
   darker.  We will be increasing the Ab incubation time to se=f that
   will help.
   

   
As  for  the Pan Keratin, we cannot get it = work at all. We use
   Protease  2  on  our Ultra stainer for FFPE tissues in=stology, but
   this  stain in MOHS is placed on fresh, unfixed tissue... by=anual
   drop  method.  Any  time we've tried to use an enzyme retrieval, th=
   tissue looks eaten up... even if we incubate if for a minute.
   

   =ASince  the tissue is not fixed, I figured that no retrieval step
   would  be=eded, but the Pan Keratin refuses to work with or without
   retrieval.
   =A
   
For  those of you in MOHS labs that are using a manual staining me   thod  for  
Melan A and Pan Keratin, would you be willing to share your
   protoc= with us?
   

   
Thanks so much!!
   

   
Karen
   

   
K=en L. Bauer HTL/HT (ASCP) | Histology Supervisor | Pathology |
   MOHS   Lab   S=ervisor  |  Dermatology  |  Phone:  715-838-3205  |
   [9]bauer.ka...@mayo.edu<[10]mailto:bauer.ka...@mayo.edu> | Mayo Clinic
   Health  System  |  122=hipple  Street  |  Eau Claire, WI 54702 |
   [11]mayoclinichealthsystem.org
   

   =A
   
___
   
Histonet ma=ing list
   
[13]Histo=t...@lists.utsouthwestern.edu
   
[14]http://lists.utsouthwestern.edu/mailman/l=tinfo/histonet
   

   
_=
   
Histonet mailing list
   
[15]Histonet@lists.utsouthwestern.edu
   
[16]http://lists.utso=hwestern.edu/mailman/listinfo/histonet
   



 


References

   1. 3D"mailto:barbara.tibbs@accurated   2. ="mailto:bauer.ka...@mayo.edu";
   3. 3D"mailto:histonet@lists.utsouthwestern.edu   4. 
3D"mailto:barbara.tibbs@accuratediagnosticlabs.c   5. 
3D"mailto:histonet-boun...@lists.utsouthwestern.edu   6. 3D"mailto:histo   7. 
3D"m

RE: [Histonet] RE: MOHS IPs

[pruegg]

   I  agree with Barbara try fixing in cold acetone/ethanol mix even = if
   just  for  a  couple  of minutes then go directly to buffer do not dry
   after=  fix,  I  would dilute the protease 1:4 with buffer (PBS or TBS
   what  ever  u  u= se) and do it for a short time.  Enzyme digestion on
   frozen  sections  i=  s  tricky  but  fixing a little and diluting the
   enzyme  should  help  prevent  ov=  er  digestion.   How  long  are  u
   incubating  the  primary?   Are  u usin= g an enhanced labeled polymer
   detection?  HRP/Dab or AEC or Alk.P/fast= red?

   


 


   
 Original Message 
   
Subject: [Histonet] RE: MOHS = IPs
   
From: Barbara Tibbs <[1]barbara.ti...@accuratediagnosticlabs.com>
   
D= ate: Wed, October 02, 2013 5:54 am
   
To: "Bauer, Karen L." <[2]bauer.ka...@mayo.edu>,
   
"'histonet@lists.utsouthwest= ern.edu'"
   
<[3]histonet@lists.utsouthwestern.edu>
   

   
Hello Karen,
   = 

   
Back  in the old days of doing IHC manually on mostly fresh, froze   n  tissue  
I would immediately place the slide with the frozen section
   into  a=  coplin  jar  of  acetone  to  fix it. Keep the coplin jar of
   acetone  in  the  cr=  yostat.  Leave  it  in  the acetone for only 10
   minutes.  Try diluting the Pr= otease 2 - 1:1 to make it gentler so it
   doesn't  "eat"  the  tissue.  This  te= chnique worked well when I was
   doing ER/PR on frozen breast tumors. Not su= re it will work with skin
   but it's worth a try.
   

   
Barbara S. Tib= bs
   
Histology Supervisor
   
Accurate Diagnostic Labs
   
South Pl= ainfield, NJ
   
[4]barbara.ti...@accuratediagnosticlabs.com
   
732-839-3374
   
C= ell: 610-809-6508
   

   

   
_= ___
   
From:  [5]histonet-boun...@lists.utsouthwestern.edu
   <[6]histonet-bounces@lists.utsouthwestern=  .edu>  on behalf of Bauer,
   Karen L. <[7]bauer.ka...@mayo.edu>
   
Sent: Tuesday, October 01, 20= 13 5:22 PM
   
To: '[8]= histonet@lists.utsouthwestern.edu'
   
Subject: [Histonet] MOHS IPs

   
Hi all,
   

   
We  are  in the process of validating some a= ntibodies in our MOHS
   lab.  The Melan A (Mart 1) antibody is working well, = but it could be
   darker.  We will be increasing the Ab incubation time to se= e if that
   will help.
   

   
As  for  the Pan Keratin, we cannot get it = to work at all. We use
   Protease  2  on  our Ultra stainer for FFPE tissues in= Histology, but
   this  stain in MOHS is placed on fresh, unfixed tissue... by= a manual
   drop  method.  Any  time we've tried to use an enzyme retrieval, th= e
   tissue looks eaten up... even if we incubate if for a minute.
   

   =  
Since  the tissue is not fixed, I figured that no retrieval step
   would  be= needed, but the Pan Keratin refuses to work with or without
   retrieval.
   = 

   
For  those of you in MOHS labs that are using a manual staining me   thod  for  
Melan A and Pan Keratin, would you be willing to share your
   protoc= ol with us?
   

   
Thanks so much!!
   

   
Karen
   

   
K= aren L. Bauer HTL/HT (ASCP) | Histology Supervisor | Pathology |
   MOHS   Lab   S=  upervisor  |  Dermatology  |  Phone:  715-838-3205  |
   [9]bauer.ka...@mayo.edu<[10]mailto:bauer.ka...@mayo.edu> | Mayo Clinic
   Health  System  |  122=  1  Whipple  Street  |  Eau Claire, WI 54702 |
   [11]mayoclinichealthsystem.org
   

   = 

   
___
   
Histonet ma= iling list
   
[13]Histo= n...@lists.utsouthwestern.edu
   
[14]http://lists.utsouthwestern.edu/mailman/l= istinfo/histonet
   

   
_= __
   
Histonet mailing list
   
[15]Histonet@lists.utsouthwestern.edu
   
[16]http://lists.utso= uthwestern.edu/mailman/listinfo/histonet
   



 


References

   1. 3D"mailto:barbara.tibbs@accurated   2. ="mailto:bauer.ka...@mayo.edu";
   3. 3D"mailto:histonet@lists.utsouthwestern.edu   4. 
3D"mailto:barbara.tibbs@accuratediagnosticlabs.c   5. 
3D"mailto:histonet-boun...@lists.utsouthwestern.edu   6. 3D"mailto:histo   7. 
3D"mailto:Bauer.Karen   8. 3D"mailto:histonet@lists.utsouthwestern.edu";
   9. 3D"mailto:bauer.kar  10. 3D"mailto:bauer.karen@mayo  11. 
3D"http://mayoclinichealt=/
  12. 3D"http:/  13. 3D"mailto:Histonet@lists.utsouthwestern.edu";
  14. 3D"http://lists.utsouthweste=/
  15. 3D"mailto:Histonet@lists.u  16. 
file://localhost/tmp/3D"h___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

[Histonet] RE: MOHS IPs

[Barbara Tibbs]

Hello Karen,

Back in the old days of doing IHC manually on mostly fresh, frozen tissue I 
would immediately place the slide with the frozen section into a coplin jar of 
acetone to fix it.  Keep the coplin jar of acetone in the cryostat.   Leave it 
in the acetone for only 10 minutes.  Try diluting the Protease 2 - 1:1 to make 
it gentler so it doesn't "eat" the tissue.   This technique worked well when I 
was doing ER/PR on frozen breast tumors.  Not sure it will work with skin but 
it's worth a try.

Barbara S. Tibbs
Histology Supervisor
Accurate Diagnostic Labs
South Plainfield, NJ
barbara.ti...@accuratediagnosticlabs.com
732-839-3374
Cell: 610-809-6508



From: histonet-boun...@lists.utsouthwestern.edu 
 on behalf of Bauer, Karen L. 

Sent: Tuesday, October 01, 2013 5:22 PM
To: 'histonet@lists.utsouthwestern.edu'
Subject: [Histonet] MOHS IPs

Hi all,

We are in the process of validating some antibodies in our MOHS lab.  The Melan 
A (Mart 1) antibody is working well, but it could be darker.  We will be 
increasing the Ab incubation time to see if that will help.

As for the Pan Keratin, we cannot get it to work at all.  We use Protease 2 on 
our Ultra stainer for FFPE tissues in Histology, but this stain in MOHS is 
placed on fresh, unfixed tissue... by a manual drop method.  Any time we've 
tried to use an enzyme retrieval, the tissue looks eaten up... even if we 
incubate if for a minute.

Since the tissue is not fixed, I figured that no retrieval step would be 
needed, but the Pan Keratin refuses to work with or without retrieval.

For those of you in MOHS labs that are using a manual staining method for Melan 
A and Pan Keratin, would you be willing to share your protocol with us?

Thanks so much!!

Karen

Karen L. Bauer HTL/HT (ASCP) | Histology Supervisor | Pathology | MOHS Lab 
Supervisor | Dermatology | Phone: 715-838-3205 | 
bauer.ka...@mayo.edu | Mayo Clinic Health System | 
1221 Whipple Street | Eau Claire, WI 54702 | 
mayoclinichealthsystem.org


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