Re: [Histonet] Help to interview new employees for the first time

[Sheeder, Christopher via Histonet]

Hi Blanca,
Hiring has become more difficult in recent years. Most employers now only 
verify past employment. They cannot divulge any corrective actions, performance 
issues or firings.
References are hand-picked by the applicant so you don't get the whole picture 
there either.
I typically ask them about their experiences in histology. Obstacles they have 
overcome, how to handle difficult customers (angry physicians) etc.
If you can give the other techs a chance to ask the candidate questions, great, 
otherwise see what questions your techs want to know.
I fully agree with Terri Braud's response. Do not have them perform any type of 
function. That is the purpose of the probationary period.
The best interview question that was ever asked of me..."Name 12 uses for a 
pencil".
Seems silly, but it's not about the answer, it's about demonstrating your 
creative problem solving skills. (it was tough, but I came up with 12!)
Best of luck!

Christopher Sheeder, HT(ASCP)QIHC
Pathology Manager | Department of Laboratories
Seattle Children's Hospital

-Original Message-
From: Blanca Lopez 
Sent: Thursday, September 12, 2019 11:48 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Help to interview new employees for the first time

I am going to interview people for Histotech position for the first time...what 
are the best questions to ask? How do I prepare myself? what is the I need to 
know that they are the best one? What should I ask or choose? Is good to put 
them in action like cutting or staining to check on their skills or what are 
your recommendations? thank you for your help

Blanca Lopez HT (ASCP)cm
Senior Histotechnologist
UT Southwestern Medical Center
Harold C. Simmons Comprehensive Cancer Center UTSTR Biorepository Tissue Lab
6000 Harry Hines Blvd NB5.102
Dallas, Texas 75390
214-648-7598
blanca.lo...@utsouthwestern.edu<mailto:blanca.lo...@utsouthwestern.edu>





UT Southwestern


Medical Center



The future of medicine, today.


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Re: [Histonet] Help to interview new employees for the first time

[Patrick Laurie via Histonet]

I'm also a fan of having any fellow employees available ask questions.  I
have my staff limit it to histology related subjects, but my thought is if
they are going to be the ones working directly with them, it helps to have
their opinions.

Patrick Laurie(HT)ASCP QIHC

Histology Manager

Celligent Diagnostics, LLC

101 East W.T. Harris Blvd  | Suite 1212 | Charlotte, NC 28262

Work: 704-970-3300  Cell: 704-266-0869


On Fri, Sep 13, 2019 at 10:40 AM Anne Murvosh via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> I'm not sure if this is an option, but we used to melt down old blocks and
> make the HT embed and cut them. This showed us how good and quick they were
> and if they actually new what they were doing. Anne
>
> -Original Message-
> From: Blanca Lopez via Histonet 
> Sent: Thursday, September 12, 2019 11:48 AM
> To: histonet@lists.utsouthwestern.edu
> Subject: [Histonet] Help to interview new employees for the first time
>
> I am going to interview people for Histotech position for the first
> time...what are the best questions to ask? How do I prepare myself? what is
> the I need to know that they are the best one? What should I ask or choose?
> Is good to put them in action like cutting or staining to check on their
> skills or what are your recommendations? thank you for your help
>
> Blanca Lopez HT (ASCP)cm
> Senior Histotechnologist
> UT Southwestern Medical Center
> Harold C. Simmons Comprehensive Cancer Center UTSTR Biorepository Tissue
> Lab
> 6000 Harry Hines Blvd NB5.102
> Dallas, Texas 75390
> 214-648-7598
> blanca.lo...@utsouthwestern.edu<mailto:blanca.lo...@utsouthwestern.edu>
>
>
>
> 
>
> UT Southwestern
>
>
> Medical Center
>
>
>
> The future of medicine, today.
>
> ___
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Re: [Histonet] Help to interview new employees for the first time

[Anne Murvosh via Histonet]

I'm not sure if this is an option, but we used to melt down old blocks and make 
the HT embed and cut them. This showed us how good and quick they were and if 
they actually new what they were doing. Anne

-Original Message-
From: Blanca Lopez via Histonet  
Sent: Thursday, September 12, 2019 11:48 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Help to interview new employees for the first time

I am going to interview people for Histotech position for the first time...what 
are the best questions to ask? How do I prepare myself? what is the I need to 
know that they are the best one? What should I ask or choose? Is good to put 
them in action like cutting or staining to check on their skills or what are 
your recommendations? thank you for your help

Blanca Lopez HT (ASCP)cm
Senior Histotechnologist
UT Southwestern Medical Center
Harold C. Simmons Comprehensive Cancer Center UTSTR Biorepository Tissue Lab
6000 Harry Hines Blvd NB5.102
Dallas, Texas 75390
214-648-7598
blanca.lo...@utsouthwestern.edu<mailto:blanca.lo...@utsouthwestern.edu>





UT Southwestern


Medical Center



The future of medicine, today.

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Re: [Histonet] Help to interview new employees for the first time

[Erin McCarthy via Histonet]

Hi Blanca,

I think practical skills tests are a great idea. BUT I also think you need
to make sure that having someone that isn't an employee doing tasks in the
lab is a responsibility your lab would willingly take on if something were
to happen. If the candidate cut themselves and was litigious they could go
after your lab. I have not heard of it happening, but I know in this day
and age it can be a risk. Otherwise I try to ask probing questions -
usually things that cannot be answered with Yes or No. Also, I try to ask
about things you really want to know - how do they manage stress, do they
have troubleshooting experience, if so can they explain the problem and how
they solved it. Are they intuitive enough, that looking back on a
troubleshooting or stressful event can they identify what they would have
done differently? I want to make sure that the people I hire can manage
stress, and think through an issue. I also try to ask them what they feel
makes a good tech, and how they think their coworkers would describe them.
It gets them thinking about the team, not just how they see themselves.

I hope this helps!

On Thu, Sep 12, 2019 at 2:06 PM Blanca Lopez via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> I am going to interview people for Histotech position for the first
> time...what are the best questions to ask? How do I prepare myself? what is
> the I need to know that they are the best one? What should I ask or choose?
> Is good to put them in action like cutting or staining to check on their
> skills or what are your recommendations? thank you for your help
>
> Blanca Lopez HT (ASCP)cm
> Senior Histotechnologist
> UT Southwestern Medical Center
> Harold C. Simmons Comprehensive Cancer Center
> UTSTR Biorepository Tissue Lab
> 6000 Harry Hines Blvd NB5.102
> Dallas, Texas 75390
> 214-648-7598
> blanca.lo...@utsouthwestern.edu
>
>
>
> 
>
> UT Southwestern
>
>
> Medical Center
>
>
>
> The future of medicine, today.
>
> ___
> Histonet mailing list
> Histonet@lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>


-- 

Erin McCarthy, HT (ASCP)
Histology Supervisor

Tempus Labs
600 W. Chicago Ave.
Chicago IL 60654
Cell: (708)269-8610

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[Histonet] Help to interview new employees for the first time

[Blanca Lopez via Histonet]

I am going to interview people for Histotech position for the first time...what 
are the best questions to ask? How do I prepare myself? what is the I need to 
know that they are the best one? What should I ask or choose? Is good to put 
them in action like cutting or staining to check on their skills or what are 
your recommendations? thank you for your help

Blanca Lopez HT (ASCP)cm
Senior Histotechnologist
UT Southwestern Medical Center
Harold C. Simmons Comprehensive Cancer Center
UTSTR Biorepository Tissue Lab
6000 Harry Hines Blvd NB5.102
Dallas, Texas 75390
214-648-7598
blanca.lo...@utsouthwestern.edu





UT Southwestern


Medical Center



The future of medicine, today.

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[Histonet] Help with frozen spleen and liver tissue!

[Allyse Mazzarelli via Histonet]

Hi all,

Can someone please provide me with more details regarding cryosectioning of
mouse spleen and liver tissue?

Currently, I've been fixing my samples in 4% PFA (they are well fixed - I
know that will be the first question asked!), and then cryoprotect the
tissue in a series of graded sucrose solutions (15% to 30%) until they
sink.

However, when I go to place the sections on the slide from the cryostat,
they look great initially under the microscope, but once they dry they have
poor morphology, especially seen in the liver.

I've never run into this issue before, with either brain or spinal cord
which are significantly more delicate.

I've tried cutting the tissue free-floating as well to see if it was how I
was placing the sections on the slide, but nothing seems to work.

The morphology in the liver is so terribly compromised that I cannot
visualize the sinusoids properly. It is baffling that once they go onto the
slide they look okay, but 5 minutes later the tissue appears to "separate"
from itself.

Does anyone work specifically in frozen tissue sections, liver and spleen
in particular? If so, would you be able to help me figure out the best way
to generate quality specimens?

Thank you!

Regards,
Allyse Mazzarelli
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Re: [Histonet] HELP Path requesting +PAP stain control

[Terri Braud via Histonet]

On Date: Mon, 23 Jul 2018 18:31:48 -0400
From: Mary Ann 
Subject: [Histonet] Positive PAP
Mary Ann wrote "Help! My pathologist has asked that a positive patient be run 
down with our PAP stain for QC.
Point me to a reference to counter this request."

Hi Mary Ann - First of all, my sympathies.  This is the kind of craziness that 
can give a pathologist a bad name.  Secondly, what does he call a patient 
positive?  Positive for what?  LOL, JK.  In response to your question, here are 
the ONLY 2 requirements for Cytology stain QC, straight from the latest CAP 
list.  See below.  As one can see, nowhere does it require any type of patient 
control, only a documented assessment of the stain quality, on "actual case 
material"  CAPs words, not mine. Good Luck! Terri
__

**REVISED**   08/21/2017
CYP.03925   Stain AssessmentPhase I
Cytology stains are assessed at least annually to ensure their proper 
storage and acceptable quality.
NOTE: Cytology stains undergoing a daily technical quality review are exempt 
from an annual assessment.
Most stains used in the cytology laboratory are not subject to outdating, so 
that assignment of expiration dates may have no meaning.  The acceptable 
performance of such stains must be confirmed at least annually by technical 
assessment on actual case material, and as part of the evaluation of 
cytopathology cases. Where applicable, expiration dates assigned by a 
manufacturer must be observed.
Evidence of Compliance:
✓   Written procedure for stain assessment AND
✓   Records of assessment of appropriate quality of each cytology stain in 
use

CYP.04300   Daily QCPhase II
Daily QCPhase II
There are records of daily review of the technical quality of cytologic 
preparations by the pathologist or supervisory-level cytotechnologist.
NOTE:  The technical quality of cytologic preparations must be checked daily 
(on days processing occurs). This includes checking all stains for predicted 
staining characteristics each day of use. This check must include all of the 
types of preparations seen that day such as cytospins, cell blocks, and liquid 
based preparations.
If preparation and staining is performed by a different laboratory, there must 
be a procedure for the laboratory performing the preparation and staining to 
verify the acceptability of the quality of preparations and the acceptability 
of controls (if needed) before transfer.  Records of this verification must be 
readily available to the laboratory performing interpretations.  There should 
also be a mechanism for feedback from the interpreting laboratory to the 
laboratory that prepared the slides of any issues with the preparations.
_

Terri L. Braud, HT(ASCP)
Anatomic Pathology Supervisor
Laboratory
Holy Redeemer Hospital
1648 Huntingdon Pike
Meadowbrook, PA 19046
ph: 215-938-3689
fax: 215-938-3874
Care, Comfort, and Heal

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[Histonet] Help for building up Technovit 7200 and 9100 techniques

[Tina Schlüter via Histonet]

 Hello Histonet-Members

I am recently asked from my lab-coordinator to establish cutting-grinding
techniques with the technovit-resins for sectioning bones and other
hard-tissue materials.
I do not have many experiences in these techniques and I am looking for
some help. And also, where can I buy buy these resins and consumables. As
far as I know, Heraeus Kulzer has a new global distributor for this resins?
Could anybody help?

Does anybody know about trainings and courses in using these resins and
establishing the methods?

Thanks in advance for any help
Tina
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Re: [Histonet] Help with preparing slides of baby guppies

[J B via Histonet]

Jenn,

Trying to fully understand. You need someone to prepare these slides again
for you?  Great quality work, you provide the specimen?  Let me know, I
would love to learn more.

Sincerely,

JB

On Wed, May 31, 2017, 9:13 AM Dearolf, Jenn via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> Hello!
>
> My name is Jenn Dearolf, and I am a professor in the Biology Department at
> Hendrix College, a small liberal arts college in Conway, AR.  I have
> written this list before to get advice about how to prevent freezing
> artifact in small muscle samples (Thanks!), but today, I have a completely
> different need.
>
> In our Zoology course at Hendrix, we use slides that have numerous serial
> sections (7 to 10 microns thickness) of a baby guppy on them that have been
> stained with H & E.  And, a box of slides, ranging from 8 to 12 slides, is
> the entire guppy sectioned from the tip of its nose to the tip of its
> tail.  However, these slides are very old, and over the years, the mounting
> media has pulled away from the sections.  In addition, numerous slides have
> been broken or lost.
>
> Unfortunately, no one in my Department now has the necessary skills to
> produce these slides for our students.  I was wondering if anyone on the
> list knew of a facility that could produce these slides.  I think we should
> be able to provide the baby guppies.  I would just have to get approval
> from our IACUC.
>
> I also have a very old paper that discusses the steps that were necessary
> to produce the slides.  I am happy to share the methodology with anyone
> that could help us, and we could discuss what we would need to do at
> Hendrix and which steps would need to be performed at your facility.
>
> I appreciate any advice folks are willing to share.  Thanks for your time
> and consideration.
>
> Sincerely,
> Jenn
>
>
> Jennifer Dearolf, Ph.D.
> Professor and Chair
> Biology Department
> Hendrix College
> 1600 Washington Ave.
> Conway, AR 72032
> (501) 450-4530 (office)
>
>
>
>
>
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[Histonet] Help with preparing slides of baby guppies

[Dearolf, Jenn via Histonet]

Hello!

My name is Jenn Dearolf, and I am a professor in the Biology Department at 
Hendrix College, a small liberal arts college in Conway, AR.  I have written 
this list before to get advice about how to prevent freezing artifact in small 
muscle samples (Thanks!), but today, I have a completely different need.

In our Zoology course at Hendrix, we use slides that have numerous serial 
sections (7 to 10 microns thickness) of a baby guppy on them that have been 
stained with H & E.  And, a box of slides, ranging from 8 to 12 slides, is the 
entire guppy sectioned from the tip of its nose to the tip of its tail.  
However, these slides are very old, and over the years, the mounting media has 
pulled away from the sections.  In addition, numerous slides have been broken 
or lost.

Unfortunately, no one in my Department now has the necessary skills to produce 
these slides for our students.  I was wondering if anyone on the list knew of a 
facility that could produce these slides.  I think we should be able to provide 
the baby guppies.  I would just have to get approval from our IACUC.

I also have a very old paper that discusses the steps that were necessary to 
produce the slides.  I am happy to share the methodology with anyone that could 
help us, and we could discuss what we would need to do at Hendrix and which 
steps would need to be performed at your facility.

I appreciate any advice folks are willing to share.  Thanks for your time and 
consideration.

Sincerely,
Jenn


Jennifer Dearolf, Ph.D.
Professor and Chair
Biology Department
Hendrix College
1600 Washington Ave.
Conway, AR 72032
(501) 450-4530 (office)





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[Histonet] Help with eosin stain

[Nair, Indu via Histonet]

Good morning Histoneters!

I am having a problem with eosin staining getting washed away in the upgrades.
Eosin was prepared in isopropyl alcohol, followed one of the 
protocols online.

Please advice.

Thank you!

indu 

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Re: [Histonet] help!!

[Tony Henwood (SCHN) via Histonet]

You can use DAB but the issue of endogenous peroxidase will rear its ugly head.

And I suppose IF being around since 1955, when Mellors first applied the 
technique to renal tissue, it has a long history of diagnostic application that 
is hard to replace.

There are other enzymes that could be used that, not being present in human 
tissue, would not require endogenous enzyme blocking for example glucose 
oxidase. The advantage of this enzyme is that there is no endogenous glucose 
oxidase activity in mammalian tissues.

Weening, J. J., & Jennette, J. C. (2012). Historical milestones in renal 
pathology. Virchows Archiv, 461(1), 3-11.

Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Principal Scientist, the Children’s Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA

From: Allan Wang [mailto:alla...@gmail.com]
Sent: Tuesday, 18 April 2017 5:51 PM
To: Tony Henwood (SCHN)
Cc: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] help!!

Tim and Tony,

Why couldn't DAB be used on frozen sections in your example?

Allan

On Mon, Apr 17, 2017 at 9:03 PM, Tony Henwood (SCHN) via Histonet 
<histonet@lists.utsouthwestern.edu<mailto:histonet@lists.utsouthwestern.edu>> 
wrote:
Hi Bianca,
Well for most Pathology departments, Immunofluorescence (IF) is used for Renal 
and Skin biopsies; looking for human Immunoglobulin (Igs) deposition on 
basement membranes. The advantage here is not so much the fluorescence, but 
that we use unfixed frozen sections. The buffer rinse before antibody 
application, removes un-bound serum immunoglobulins, leaving any pathological 
bound Igs for the IF antibody to bind to. This gives a clean result.

If one would do IF on formalin-fixed paraffin sections of renal or skin 
biopsies, you would find heavy background due to the fixative cross-linking 
serum Igs to tissue and cells (which would usually be removed by the buffer 
rinse if unfixed frozen sections were used - see above).

IF, apart from being a historic method, also does not suffer from endogenous 
peroxidase that would need to be blocked if peroxidase was used in place of 
fluorescence.



Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA

-Original Message-
From: Blanca Lopez via Histonet 
[mailto:histonet@lists.utsouthwestern.edu<mailto:histonet@lists.utsouthwestern.edu>]
Sent: Thursday, 13 April 2017 11:10 PM
To: histonet@lists.utsouthwestern.edu<mailto:histonet@lists.utsouthwestern.edu>
Subject: [Histonet] help!!
Hello!
I just need a help with a simple question...Is anyone can explain me what is 
the purpose between performing immunohistochemistry and Immunofluorescence?
Thanks  :)

Blanca Lopez
Histotech (ASCP)
UTSW Tissue Resource K1.210
Simmons Comprehensive Cancer Center
UT Southwestern Medical Center
Telephone: 214-648-7598
Email: blanca.lo...@utsouthwestern.edu<mailto:blanca.lo...@utsouthwestern.edu>




UT Southwestern


Medical Center



The future of medicine, today.

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Re: [Histonet] help!!

[Allan Wang via Histonet]

Tim and Tony,

Why couldn't DAB be used on frozen sections in your example?

Allan

On Mon, Apr 17, 2017 at 9:03 PM, Tony Henwood (SCHN) via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> Hi Bianca,
> Well for most Pathology departments, Immunofluorescence (IF) is used for
> Renal and Skin biopsies; looking for human Immunoglobulin (Igs) deposition
> on basement membranes. The advantage here is not so much the fluorescence,
> but that we use unfixed frozen sections. The buffer rinse before antibody
> application, removes un-bound serum immunoglobulins, leaving any
> pathological bound Igs for the IF antibody to bind to. This gives a clean
> result.
>
> If one would do IF on formalin-fixed paraffin sections of renal or skin
> biopsies, you would find heavy background due to the fixative cross-linking
> serum Igs to tissue and cells (which would usually be removed by the buffer
> rinse if unfixed frozen sections were used - see above).
>
> IF, apart from being a historic method, also does not suffer from
> endogenous peroxidase that would need to be blocked if peroxidase was used
> in place of fluorescence.
>
>
>
> Regards
> Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
> Principal Scientist, the Children's Hospital at Westmead
> Adjunct Fellow, School of Medicine, University of Western Sydney
> Tel: 612 9845 3306
> Fax: 612 9845 3318
> Pathology Department
> the children's hospital at westmead
> Cnr Hawkesbury Road and Hainsworth Street, Westmead
> Locked Bag 4001, Westmead NSW 2145, AUSTRALIA
>
> -Original Message-
> From: Blanca Lopez via Histonet [mailto:histonet@lists.utsouthwestern.edu]
> Sent: Thursday, 13 April 2017 11:10 PM
> To: histonet@lists.utsouthwestern.edu
> Subject: [Histonet] help!!
>
> Hello!
> I just need a help with a simple question...Is anyone can explain me what
> is the purpose between performing immunohistochemistry and
> Immunofluorescence?
> Thanks  :)
>
> Blanca Lopez
> Histotech (ASCP)
> UTSW Tissue Resource K1.210
> Simmons Comprehensive Cancer Center
> UT Southwestern Medical Center
> Telephone: 214-648-7598
> Email: blanca.lo...@utsouthwestern.edu
>
>
> 
>
> UT Southwestern
>
>
> Medical Center
>
>
>
> The future of medicine, today.
>
> ___
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Re: [Histonet] help!!

[Tony Henwood (SCHN) via Histonet]

Hi Bianca,
Well for most Pathology departments, Immunofluorescence (IF) is used for Renal 
and Skin biopsies; looking for human Immunoglobulin (Igs) deposition on 
basement membranes. The advantage here is not so much the fluorescence, but 
that we use unfixed frozen sections. The buffer rinse before antibody 
application, removes un-bound serum immunoglobulins, leaving any pathological 
bound Igs for the IF antibody to bind to. This gives a clean result.

If one would do IF on formalin-fixed paraffin sections of renal or skin 
biopsies, you would find heavy background due to the fixative cross-linking 
serum Igs to tissue and cells (which would usually be removed by the buffer 
rinse if unfixed frozen sections were used - see above).

IF, apart from being a historic method, also does not suffer from endogenous 
peroxidase that would need to be blocked if peroxidase was used in place of 
fluorescence.



Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-Original Message-
From: Blanca Lopez via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Thursday, 13 April 2017 11:10 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] help!!

Hello!
I just need a help with a simple question...Is anyone can explain me what is 
the purpose between performing immunohistochemistry and Immunofluorescence?
Thanks  :)

Blanca Lopez
Histotech (ASCP)
UTSW Tissue Resource K1.210
Simmons Comprehensive Cancer Center
UT Southwestern Medical Center
Telephone: 214-648-7598
Email: blanca.lo...@utsouthwestern.edu




UT Southwestern


Medical Center



The future of medicine, today.

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Re: [Histonet] help!!

[Caroline Miller via Histonet]

Blanca,
Here are my feelings on this, and I am sure a lot of other folks have feels
here too, so please chime in.

1 - I feel that most clinical labs are more on the IHC bandwagon and
research labs are IF (with the exception of IgG staining in kidney biopsies
or bullous disease in skin- which is because the antibodies don't like
formalin fixing (if this is now wrong I am sorry, I haven't been in a
clinical lab in quite a while). Research labs are often also working with
genetically encoded fluorophores such as GFP, YFP, mCherry
2 - Formalin fixation (especially over fixation) can often lead to a large
amount of autofluorescence in the 488 region, which is a common place for
secondary antibodies and also GFP. Research labs have a lot more control
over their fixation protocols.
3 - The microscopes commonly available to clinical labs are bright field
scopes and in research labs fluorescent scopes
4 - Fluorescence can provide more contrast to a positively localized
fluorophore, but sometimes at the detriment of viewing the overall
morphology of the tissue like you get with bright field IHC and a nuclear
counterstain.
5 - Research lab protocols are often very 'experimental' and can lead to
increased tissue damage, which again is not viewed under the fluorescence
microscope (as much). Clinical labs have lots of experience and also
defined protocols that work well in the IHC / bright field space.
6 - the only real difference is the detection method, you can use any
primary antibody with either ABC/ impress / enzyme based methods or with
fluorophore conjugated secondaries.

So, in short - no *real* reason, but mainly that is the way things shook
out.

I could go on about researchers not understanding how to take photos on a
bright field scopes too, but that is too broad a statement, but as a core
director I saw them being more comfortable with the fluorescent methods :)

mills




On Thu, Apr 13, 2017 at 6:09 AM, Blanca Lopez via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> Hello!
> I just need a help with a simple question...Is anyone can explain me what
> is the purpose between performing immunohistochemistry and
> Immunofluorescence?
> Thanks  :)
>
> Blanca Lopez
> Histotech (ASCP)
> UTSW Tissue Resource K1.210
> Simmons Comprehensive Cancer Center
> UT Southwestern Medical Center
> Telephone: 214-648-7598
> Email: blanca.lo...@utsouthwestern.edu
>
>
> 
>
> UT Southwestern
>
>
> Medical Center
>
>
>
> The future of medicine, today.
>
> ___
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>



-- 
Caroline Miller (mills)
Director of Histology
3Scan.com
415 2187297
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Re: [Histonet] help!!

[Morken, Timothy via Histonet]

Blanca, immunofluorescence (IF) is a subset of immunochemistry. 
Immunohistochemistry is also a subset of immunochemistry. There is some overlap 
between the two.

Immunohistochemistry denotes  immunochemistry done on tissue sections 
("-histo-" =" tissue"). But we can also use other enzymes to label the 
antibodies for immunohistochemistry (peroxidase, alkaline phosphatase, etc).


IF is just one of many methods of labeling the antibodies with a visual label. 
Others are peroxidase and alkaline phosphatase.


Generally IF is done on "fresh" cells or tissue. For tissue it is normally 
frozen tissue. 

IF can be done on cells (ie, immunocytochemistry) either on slides (smears, 
various preparations) or in solution as with flow cytometry - the cells are 
labeled with fluorescent-labeled antibodies and sorted by color (or no color). 

Generally the IF method is faster to perform because there is no processing 
beyond freezing the tissue. In the past IF was also more sensitive due to dark 
field microscopy in the fluorescence microscope. With the advent of various 
methods to amplify the signal (avidin -biotin, polymers with multiple enzymes) 
the peroxidase methods are just as sensitive, if not more so.

But fresh or frozen tissue has the advantage of the epitopes remaining unfixed, 
especially by formalin - which can mask the antigen from the antibody. Some 
antibodies do not work well on formalin-fixed tissue, even if antigen retrieval 
is used, so frozen tissue or cells are the best option. 



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





-Original Message-
From: Blanca Lopez via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Thursday, April 13, 2017 6:10 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] help!!

Hello!
I just need a help with a simple question...Is anyone can explain me what is 
the purpose between performing immunohistochemistry and Immunofluorescence?
Thanks  :)

Blanca Lopez
Histotech (ASCP)
UTSW Tissue Resource K1.210
Simmons Comprehensive Cancer Center
UT Southwestern Medical Center
Telephone: 214-648-7598
Email: blanca.lo...@utsouthwestern.edu




UT Southwestern


Medical Center



The future of medicine, today.

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[Histonet] help!!

[Blanca Lopez via Histonet]

Hello!
I just need a help with a simple question...Is anyone can explain me what is 
the purpose between performing immunohistochemistry and Immunofluorescence?
Thanks  :)

Blanca Lopez
Histotech (ASCP)
UTSW Tissue Resource K1.210
Simmons Comprehensive Cancer Center
UT Southwestern Medical Center
Telephone: 214-648-7598
Email: blanca.lo...@utsouthwestern.edu




UT Southwestern


Medical Center



The future of medicine, today.

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[Histonet] Help with CryoJane Bone cutting

[Owens, Katrina via Histonet]

Hello All:

I recently acquired a CryoJane unit but it doesn't seem to work in regards to 
bone cutting. It works just fine for soft tissue, but getting the bone to 
transfer from the tape to the slide just isn't happening. I have tried, 
successfully, using other UV sources to transfer the bone from the tape to the 
slides, but just not with the CryoJane.

Any help or advice in this matter would be greatly appreciated.

Thank you and have a good day!

Katrina Owens
owen...@ccf.org

===


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[Histonet] Help with liver/heart tissues

[Allyse Mazzarelli via Histonet]

Good afternoon,

I am having a few issues getting nice morphology from both liver and heart
tissues. I currently work only with CNS tissues (brain, spinal cord, DRG,
etc.) but recently, our research team have become interested in
immunostaining some peripheral tissues, including the heart and liver.

All tissue [mouse] is perfusion fixed with 4% PFA and then post-fixed in
fresh 4% PFA for at least 24 - 48 hours. After fixation, the tissue is
cryoprotected in 30% sucrose solution for 48 hours. Once cryoprotected, I
then section the tissue at various thicknesses depending on the assay I'm
running.

Previously I sectioned some heart and liver samples (sectioned both at 20um
and at 8um) and ran a few H Unfortunately, the tissue was so damaged
and under-fixed that we scrapped the blocks.

This time around, the mouse liver tissue was carefully dissected prior to
post-fixation into four quadrants to allow for better PFA permealization.
Additionally, we post fixed in 4% PFA for 3 days instead of 2, and the
tissue was cryoprotected for 2 days.

To my surprise, when I sectioned this tissue on the cryostat, I still
noticed severe artifacts. It is very difficult to see nice morphology, and
there appears that there was an issue with fixation (in the liver the
nucleus is flattened and the sinusoids are not clear, etc. the nuclei in
the heart are also more flat and the muscle fibers have separated from one
another). The staining was not as bad as the first tissue I had sectioned,
but I was still unable to get a nice H stain depicting clear
nuclear/cytoplasm. These artifacts appeared at both 8um and 20um.

Does anyone happen to have nice fixation/H staining protocols for both
liver and heart? I'd be happy to give an in-depth description of the
protocol(s) I'm currently using. Additionally, is there anything that
appears I'm doing wrong in terms of perfusion/fixation?

Thanks so much!

Allyse
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[Histonet] Help on Competency Assessments

[Fawn Bomar via Histonet]

Hey everyone,



I know that this question has been asked before and I have tried to find out 
information through the internet, but I am having a difficult time trying to 
find any type of sample forms.  Would anyone be willing to share their 
competency assessment forms/checklists?  I am trying to make one up for our lab 
and different areas and need some guidance to ensure that I cover everything 
that needs to be assessed.  I know that I need to have the collection and 
transporting of specimens from the OR/Endo areas, receiving/accessioning and 
labeling specimens/blocks/slides.  Preparing/sectioning/staining Frozens.  
Embedding/sectioning/staining routine stains/special stains/IHC stains.  
Documenting/performing of equipment maintenance/function 
checks/decons/temperature checks.  Preparing and labeling cytology specimens 
received in lab/ prepared FNA slides/specimens from FNA's/ Prepared 
slides/specimens from CT guided procedures.



I am having a hard time trying to figure out exactly how to document and which 
of the six (if not all) competency elements are needed to be checked for each 
of the areas.



If anyone is willing to share their templates (and how they actually document, 
i.e., initial, checkmark)  or perhaps point me to a website, book, or person to 
reference, I would be very grateful!!!



Thank you,



Fawn Bomar
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Re: [Histonet] Help with causes or theory behind failure of nuclear counterstaining after IHC!

[WILLIAM DESALVO via Histonet]

Maria, I. Think you may have a pH issue. High pH results in reduction of 
protons, H+, effects dye structure and can cause light to no staining after 
bluing. If you are using hematoxylin w/ aluminum, most popular, decreased pH = 
decreased intensity. Acid breaks the Al+3. Check your ph throughout the 
process. Sounds like something has changed. Good luck.

Sent from my Windows Phone

From: Morken, Timothy via Histonet<mailto:histonet@lists.utsouthwestern.edu>
Sent: ‎1/‎4/‎2016 9:20 AM
To: Maria Mejia<mailto:mbmph...@gmail.com>
Cc: Histonet<mailto:histonet@lists.utsouthwestern.edu>
Subject: Re: [Histonet] Help with causes or theory behind failure of nuclear 
counterstaining after IHC!

Maria,

If the counterstain is good when done before IHC stain and poor after it sounds 
like proteins are being extracted during the IHC processing and staining. Have 
you tried staining sections after each step of the IHC process to isolate the 
point the stain becomes weak?

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



-Original Message-
From: Maria Mejia via Histonet [mailto:histonet@lists.utsouthwestern.edu]
Sent: Sunday, January 03, 2016 8:29 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Help with causes or theory behind failure of nuclear 
counterstaining after IHC!

Happy New Year Everyone,

I'm the lead histologist working in an IHC based research lab focused on early 
stages of Alzheimer's disease.  We work on paraffin sections processed & cut 
from 600um celloidin sections.  Including a lot of 60um cellodin sections from 
whole human brainstem.

For years, everything has going good regarding counterstaining after single & 
double IHC staining on 60um free-floating sections.  However for the past two 
months we've struggled to achieve good visible counterstaining on IHC sections 
to count the stained neurons - to see clearly the nucleus & nucleolus!

For a number of years, Gallocyanine was our choice of counterstain after IHC.  
Now, it's NOT working (neurons not stained visible enough to count).  We've 
also tried cresyl violet
counterstain -  staining too weak!   In both counterstains, we modified
the staining protocols quite a number of times to get good visible staining - 
nothing!!!

Strangle because we get lovely counterstained neurons with NO IHC staining on 
our 60um sections, but as soon as we take the sections through the IHC protocol 
e.g. antigen retrieval, antibodies & chromogens - counterstain is too weak!  
Our paraffin IHC sections work & look wonderful!

Now, my PI wants to try methyl green counterstain, however I think we'll have 
the same problem.
Here's what I need help with:

1) Can someone please explain the reason or theory  behind the failure of 
counterstain uptake by cells such as human neurons on 60um celloidin sections?

2) Can anyone please offer staining protocols that use alternative dehydration 
& clearing reagents.
I've been using alcohols dehydration (96% & 100%) without success as well as 
clearing with xylene - which hardens the tissue if left too long in this 
reagent.

 I was thinking of perhaps using acetone instead of alcohols & maybe using a 
methyl salicylate or chloroform.  Thoughts anyone?

I wish Dr Chris van der Loos was still with us.  I'd dearly like to hear from 
anyone who can help with this issue - Gayle Callis, Terry Johnson, Dr Hohn 
Kiernan et al.

Any assistance anyone can provide will be greatly appreciated!

Best
Maria Mejia
UCSF
Memory & Aging Department
San Francisco, CA
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[Histonet] Help with causes or theory behind failure of nuclear counterstaining after IHC! (Maria Mejia)

[Steven Weston via Histonet]

Maria,
This may sound simplistic but I find that if I have problems such as this the 
first thing I do is try a new batch of the staining reagents. If you haven't 
had problems before then something must have changed either in your protocols 
or your reagents. It could be that your heat retrieval reagents are too old or 
contain detergents that remove some of the proteins you are looking for. Some 
of the proprietary heat retrieval reagents that allow you to heat retrieve 
without having to dewax  by going through xylene have been shown to change the 
nuclear staining pattern and create what appear to be nuclei that are full of 
vacuoles.
Also if the celloidion is not completely removed during your staining it may be 
stopping any of the higher molecular weight stains from penetrating the cells. 
Try leaving your sections in acetone for a number of changes to ensure full 
removal of the celloidion.
Regards
Steve Weston
University of Tasmania
Breathe-Well CRE
Lab Manager
0408990859




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Re: [Histonet] Help with causes or theory behind failure of nuclear counterstaining after IHC!

[Morken, Timothy via Histonet]

Maria, 

If the counterstain is good when done before IHC stain and poor after it sounds 
like proteins are being extracted during the IHC processing and staining. Have 
you tried staining sections after each step of the IHC process to isolate the 
point the stain becomes weak? 

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



-Original Message-
From: Maria Mejia via Histonet [mailto:histonet@lists.utsouthwestern.edu] 
Sent: Sunday, January 03, 2016 8:29 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Help with causes or theory behind failure of nuclear 
counterstaining after IHC!

Happy New Year Everyone,

I'm the lead histologist working in an IHC based research lab focused on early 
stages of Alzheimer's disease.  We work on paraffin sections processed & cut 
from 600um celloidin sections.  Including a lot of 60um cellodin sections from 
whole human brainstem.  

For years, everything has going good regarding counterstaining after single & 
double IHC staining on 60um free-floating sections.  However for the past two 
months we've struggled to achieve good visible counterstaining on IHC sections 
to count the stained neurons - to see clearly the nucleus & nucleolus!  

For a number of years, Gallocyanine was our choice of counterstain after IHC.  
Now, it's NOT working (neurons not stained visible enough to count).  We've 
also tried cresyl violet
counterstain -  staining too weak!   In both counterstains, we modified
the staining protocols quite a number of times to get good visible staining - 
nothing!!!

Strangle because we get lovely counterstained neurons with NO IHC staining on 
our 60um sections, but as soon as we take the sections through the IHC protocol 
e.g. antigen retrieval, antibodies & chromogens - counterstain is too weak!  
Our paraffin IHC sections work & look wonderful!

Now, my PI wants to try methyl green counterstain, however I think we'll have 
the same problem.
Here's what I need help with:

1) Can someone please explain the reason or theory  behind the failure of 
counterstain uptake by cells such as human neurons on 60um celloidin sections?

2) Can anyone please offer staining protocols that use alternative dehydration 
& clearing reagents.
I've been using alcohols dehydration (96% & 100%) without success as well as 
clearing with xylene - which hardens the tissue if left too long in this 
reagent. 

 I was thinking of perhaps using acetone instead of alcohols & maybe using a 
methyl salicylate or chloroform.  Thoughts anyone?

I wish Dr Chris van der Loos was still with us.  I'd dearly like to hear from 
anyone who can help with this issue - Gayle Callis, Terry Johnson, Dr Hohn 
Kiernan et al.

Any assistance anyone can provide will be greatly appreciated!

Best
Maria Mejia
UCSF
Memory & Aging Department
San Francisco, CA
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[Histonet] Help with causes or theory behind failure of nuclear counterstaining after IHC!

[Maria Mejia via Histonet]

Happy New Year Everyone,

I'm the lead histologist working in an IHC based research lab focused on early 
stages
of Alzheimer's disease.  We work on paraffin sections processed & cut from 
600um celloidin 
sections.  Including a lot of 60um cellodin sections from whole human 
brainstem.  

For years, everything has going good regarding counterstaining after single & 
double IHC staining
on 60um free-floating sections.  However for the past two months we've 
struggled to achieve
good visible counterstaining on IHC sections to count the stained neurons - to 
see clearly
the nucleus & nucleolus!  

For a number of years, Gallocyanine was our choice of counterstain after IHC.  
Now, it's
NOT working (neurons not stained visible enough to count).  We've also tried 
cresyl violet
counterstain -  staining too weak!   In both counterstains, we modified
the staining protocols quite a number of times to get good visible staining - 
nothing!!!

Strangle because we get lovely counterstained neurons with NO IHC staining on 
our 60um
sections, but as soon as we take the sections through the IHC protocol e.g. 
antigen retrieval, 
antibodies & chromogens - counterstain is too weak!  Our paraffin IHC sections 
work & look 
wonderful!

Now, my PI wants to try methyl green counterstain, however I think we'll have 
the same problem.
Here's what I need help with:

1) Can someone please explain the reason or theory  behind the failure of 
counterstain uptake
by cells such as human neurons on 60um celloidin sections?

2) Can anyone please offer staining protocols that use alternative dehydration 
& clearing reagents.
I've been using alcohols dehydration (96% & 100%) without success as well as 
clearing with xylene - 
which hardens the tissue if left too long in this reagent. 

 I was thinking of perhaps using acetone instead of alcohols & maybe using a 
methyl salicylate or
chloroform.  Thoughts anyone?

I wish Dr Chris van der Loos was still with us.  I'd dearly like to hear from 
anyone who can help with
this issue - Gayle Callis, Terry Johnson, Dr Hohn Kiernan et al.

Any assistance anyone can provide will be greatly appreciated!

Best
Maria Mejia
UCSF
Memory & Aging Department
San Francisco, CA
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[Histonet] Help: IHC service

[Karen Cai via Histonet]

HELP:

 

Hi,

Is there anybody can provide me the price list/structure of the custom IHC
services? 

 

For example, to test 200 tissue slides and get 200 images, how much does it
cost?

 

Thank you very much in advance,

 

Have a nice weekend,

 

Best Regards,

Karen

k...@prosci-inc.com

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Re: [Histonet] Help: IHC service

[Colleen Forster via Histonet]

I'd be very interested in this as well...could you shaere Karen.


Thanks.


C

On Mon, Nov 2, 2015 at 11:43 AM, Karen Cai via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> HELP:
>
>
>
> Hi,
>
> Is there anybody can provide me the price list/structure of the custom IHC
> services?
>
>
>
> For example, to test 200 tissue slides and get 200 images, how much does it
> cost?
>
>
>
> Thank you very much in advance,
>
>
>
> Have a nice weekend,
>
>
>
> Best Regards,
>
> Karen
>
> k...@prosci-inc.com
>
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[Histonet] Help with "Quotes" for presentation

[james mcmurry via Histonet]

Hello Fellow Histo techs,
This is my first time posting to Histonet. I am looking for experienced 
individuals to provide me with a quote (very short 1-2 sentences) on what you 
feel has changed most in histology over the years. I am presenting at the MSH 
fall symposium and as part of my presentation I would like to include several 
slides of quotes from Histotechs. My presentation is called "A walk down memory 
lane". I am a brand new tech just getting her feet wet and wanted to talk to 
other about the changes in histology. Until I started talking to some techs 
with 20-30+ years experience I never knew how things were done years ago. I 
found it very interesting to say the least. Here is a very general example on 
what I am looking for but please use this similair format:

For me the greatest change is histology has been the increase in personalized 
care. Next-Gen Sequencing is really changing how we treat a patients exact 
cancer.
Lisa M. 
McMurry HT (Henry Ford Health System, Detroit-MI)

It could be on microtomes, embedding, special stains, tech duties, molecular or 
just anything histology related. I am hoping to get 4-6 good quotes. I hope you 
find a minute to shoot me back a email. Thank you histo buddies for all your 
help!!

Lisa McMurry BS, LVT, HT
jrmelect...@att.net


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[Histonet] Help with PowerPath -- how do you use it?

[Cheryl]

 Hello All,

I need help -- is anyone using PowerPath with the Amp module for barcode and
driving slide label production?  I am hoping for a 5-10 minute conversation
on your workflow, your worksheets, what is working and where you've had to
come up with solutions (where it doesn't work as well as you'd hoped).

I have to make a couple of decisions for my hospital employer and don't know
enough to make them! 

Call me, write me back on here or in private:

My cell 281.883.7704
Email tkngfl...@yahoo.com

Thank you!

Cheryl Kerry


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[Histonet] Help with a survey on research antibodies

[Chiriboga, Luis]

Dear Colleagues,

You are all well  aware of the reproducibility issues when using antibodies for 
IHC. I'm working with the Global Biological Standards Institute (GBSI) to 
address two important issues-1) cell culture authentication practices and 2) 
research antibodies.  I have agreed to help GBSI distribute the surveys through 
this link http://surveys.mckinley-advisors.com/s3/LC to gather valuable 
information about how researchers view the best practices in each of these 
areas and what they see as the challenges and barriers to implementing those 
practices.  GBSI will be analyzing the data and publishing the results later in 
2015.  I strongly encourage you to participate or to distribute to your staff 
for their participation.

At the beginning of the survey (question #3), you will notice that there is a 
simple way to take one or both surveys. GBSI's pretesting indicates that each 
survey can be completed in as little as 10 minutes.

Please contact Mark Gibson at GBSI, mgib...@gbsi.orgmailto:mgib...@gbsi.org 
if you have specific questions about the survey instrument.

Best  regards and thanks for your time

Luis


Luis Chiriboga Ph.D, Director
OCS Experimental Pathology IHC Core Lab
NYU School of Medicine
Smilow 308/310
Office: 646-501-6934
Mobile: (347) 712-0897
luis.chirib...@nyumc.orgmailto:luis.chirib...@nyumc.org

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[Histonet] Help with cutting mouse brain at 9-10 microns

[Coffey, Anna (NIH/NCI) [C]]

Hi Kimberly,

Most of what I work with is mouse tissue and I've found the brains to be a bit 
tricky because they both hydrate and dry out quickly. I normally keep the 
paraffin blocks on an ice for about 2 hours (after they've been fully faced 
in), checking periodically to make sure the tissue is not overhydrating. When I 
section, I can normally only take a few sections before the brain starts to dry 
out again (you can tell when you start to see scratches and dry white areas on 
the tissue). Most of the blocks are ready to cut again after a few additional 
minutes back on the ice.

For thicker sections (up to 20um), I take use the wooden stick of a cotton swab 
and hold it against the base of the paraffin block as I cut the section. The 
section will curl around the stick and you can roll it out flat on the water 
bath to smooth it out.

Hope this helps!

Anna Coffey, MS, HTL(ASCP)CM
Histotechnologist
Center for Advanced Preclinical Research
Frederick National Laboratory for Cancer Research
Leidos Biomedical Research, Inc.
Bld 539, 224
Frederick, Maryland 21702
301-846-1730

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[Histonet] Help with cutting mouse brain at 9-10 Microns

[Kimberly Marshall]

?Hello Histo folks


  I am starting a research project with mouse brain,  I am having trouble with 
chatter on the regular 3.5 mm sections and cant get the 9-10 mm to cut at all.  
I have soaked them in warm water and wonder in using a  softener like 
conditioner would help.  I am new to the animal tissue world and any advise 
would help.


Thanks in advance.

Kimberly
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Re: [Histonet] HELP! Need some old fashioned histology advice

[Jay Lundgren]

 I don't know if the slides were subbed or not, but they looked
improvised.  The point is, at some point the sections are going to have to
be mounted for visualisation, right?  Someone or something is going to look
at the section under magnification?

 If you mount them on glass *before* staining, the whole problem of
batching becomes one of improvising a giant stain rack and giant staining
line.  Don't forget, you need giant cover slips, giant slide folders, and
giant postdocs.
I know that lab glassware is custom ordered every day.  Sounds expensive
and time consuming to set up, but it beats fiddling around with free
floating tissue anyday, IMHO.

I don't know what the Ab penetration is going to be like on a 100um
section, because they weren't using IHC at Genentech, they were doing in
vitro DNA hybridization on the slides.  I'm assuming the section thickness
is necessary because the PI wants to follow axonal pathways and see the
patterns of staining?  Sounds like a fun project.  If you need more help
later you can PM me.

   Sincerely,

  Jay A. Lundgren,
M.S., HTL (ASCP)

On Mon, Jan 5, 2015 at 10:38 PM, Mejia, Mary mary.me...@ucsf.edu wrote:

  Hello Patsy,

  Thank you very much for responding!  Yes  of course, I'll be removing
 the celloidin from each section - we normally
 do this on smaller human whole brainstem sections using ether/100% EA 1:1
 - 3x - 3 minutes each with agitation.  The
 latter type of sections are very easy to work with, however these future
 big boys I'll be getting is another thing.

  Our lab is currently alcohol processing a human whole brain  after the
 last 95% EA - it will be embedded in 2% celloidin
  placed inside a very large desiccator under 20 psi pressure.  This part
 like every step will take some period of time,
 I need to test several different IHC methods  hope one will actually work.

  If you have any further ideas or thoughts on this subject - shoot me an
 email.  Thank you again for responding.

  Maria
  --
 *From:* Patsy Ruegg [prueg...@hotmail.com]
 *Sent:* Sunday, January 04, 2015 7:03 PM
 *To:* Jay Lundgren; Maria Mejia
 *Cc:* Histonet@Lists. Edu; Mejia, Mary
 *Subject:* RE: [Histonet] HELP! Need some old fashioned histology advice

   I have done something similar to this but I used tissue that was fixed
 but not processed and embedded, this is called enblock labeling, I
 infiltrated the fixed tissue with the IHC reagents, in a vial/tube, the
 blocking reagents, then the antibody, then the detection reagents and DAB,
 then dehydrated the tissue.  I used vials or tubes on a platform shaker and
 would infiltrate reagents for days, then after it was done I infiltrated
 and embedded the tissue in glycol methacrylate (GMA) so that I could
 section it, it actually worked.  The tissue was already IHc LABeled so all
 I did to the 5 micron sections after they were cut was a hematoxylin
 counterstain, this was mineralized bone so I had to embedd in something
 hard like GMA to section.

 Will you remove the Celloidin before trying to do the IHC staining?  100
 micron sections might be easy to float/handle using a glass pipette for
 transferring.  Sounds like an interesting project, good luck and feel free
 to ask for advise and keep us posted on your progress.

 Cheers,
 Patsy

 Patsy Ruegg, HT(ASCP)QIHC
 Ruegg IHC Consulting
 40864 E Arkansas Ave
 Bennett, CO 80102
 H 303-644-4538
 C 720-281-5406
 prueg...@hotmail.com



  Date: Sun, 4 Jan 2015 11:58:38 -0600
  From: jaylundg...@gmail.com
  To: mbmph...@gmail.com
  Subject: Re: [Histonet] HELP! Need some old fashioned histology advice
  CC: histonet@lists.utsouthwestern.edu; mary.me...@ucsf.edu
 
  I can help with the old fashioned advice:
 
 
  - 1 scant teaspoon simple syrup
  - 2 dashes Angostura Bitters, plus more to taste
  - 1 half dollar–sized slice orange peel, including pith
  - 2 ounces good-quality rye or bourbon
  - 1 maraschino cherry
 
  As for the Histology, is there any reason you cannot mount the
  sections onto glass slides? When I was working at Genentech they were
  cutting frozen sections through whole rabbits and mounting the sections
 on
  (giant) glass slides. I think that rolling the tissue up, inserting it,
  and then removing it from a glass tube would destroy the tissue.
 
  Sincerely,
 
  Jay A. Lundgren, M.S., HTL
  (ASCP)
 
  On Sat, Jan 3, 2015 at 8:01 PM, Maria Mejia mbmph...@gmail.com wrote:
 
   First, the very best of holidays to everyone.
  
   Now for the histology part. Our lab's focus is on the early stages of
   Alzheimer's Disease in the Brainstem
   using celloidin processing  embedding for IHC staining. This year, our
   lab will be receiving 6 post-mortem
   whole human brains (1 every other month). After fixation, processing 
   celloidin embedding, the whole brain
   will be serially cut at 100um thick. Each brain section will be 5

RE: [Histonet] HELP! Need some old fashioned histology advice

[Mejia, Mary]

Hello Patsy,

Thank you very much for responding!  Yes  of course, I'll be removing the 
celloidin from each section - we normally
do this on smaller human whole brainstem sections using ether/100% EA 1:1 - 3x 
- 3 minutes each with agitation.  The
latter type of sections are very easy to work with, however these future big 
boys I'll be getting is another thing.

Our lab is currently alcohol processing a human whole brain  after the last 
95% EA - it will be embedded in 2% celloidin
 placed inside a very large desiccator under 20 psi pressure.  This part like 
every step will take some period of time,
I need to test several different IHC methods  hope one will actually work.

If you have any further ideas or thoughts on this subject - shoot me an email.  
Thank you again for responding.

Maria

From: Patsy Ruegg [prueg...@hotmail.com]
Sent: Sunday, January 04, 2015 7:03 PM
To: Jay Lundgren; Maria Mejia
Cc: Histonet@Lists. Edu; Mejia, Mary
Subject: RE: [Histonet] HELP! Need some old fashioned histology advice

I have done something similar to this but I used tissue that was fixed but not 
processed and embedded, this is called enblock labeling, I infiltrated the 
fixed tissue with the IHC reagents, in a vial/tube, the blocking reagents, then 
the antibody, then the detection reagents and DAB, then dehydrated the tissue.  
I used vials or tubes on a platform shaker and would infiltrate reagents for 
days, then after it was done I infiltrated and embedded the tissue in glycol 
methacrylate (GMA) so that I could section it, it actually worked.  The tissue 
was already IHc LABeled so all I did to the 5 micron sections after they were 
cut was a hematoxylin counterstain, this was mineralized bone so I had to 
embedd in something hard like GMA to section.

Will you remove the Celloidin before trying to do the IHC staining?  100 micron 
sections might be easy to float/handle using a glass pipette for transferring.  
Sounds like an interesting project, good luck and feel free to ask for advise 
and keep us posted on your progress.

Cheers,
Patsy

Patsy Ruegg, HT(ASCP)QIHC
Ruegg IHC Consulting
40864 E Arkansas Ave
Bennett, CO 80102
H 303-644-4538
C 720-281-5406
prueg...@hotmail.com



 Date: Sun, 4 Jan 2015 11:58:38 -0600
 From: jaylundg...@gmail.com
 To: mbmph...@gmail.com
 Subject: Re: [Histonet] HELP! Need some old fashioned histology advice
 CC: histonet@lists.utsouthwestern.edu; mary.me...@ucsf.edu

 I can help with the old fashioned advice:


 - 1 scant teaspoon simple syrup
 - 2 dashes Angostura Bitters, plus more to taste
 - 1 half dollar–sized slice orange peel, including pith
 - 2 ounces good-quality rye or bourbon
 - 1 maraschino cherry

 As for the Histology, is there any reason you cannot mount the
 sections onto glass slides? When I was working at Genentech they were
 cutting frozen sections through whole rabbits and mounting the sections on
 (giant) glass slides. I think that rolling the tissue up, inserting it,
 and then removing it from a glass tube would destroy the tissue.

 Sincerely,

 Jay A. Lundgren, M.S., HTL
 (ASCP)

 On Sat, Jan 3, 2015 at 8:01 PM, Maria Mejia mbmph...@gmail.com wrote:

  First, the very best of holidays to everyone.
 
  Now for the histology part. Our lab's focus is on the early stages of
  Alzheimer's Disease in the Brainstem
  using celloidin processing  embedding for IHC staining. This year, our
  lab will be receiving 6 post-mortem
  whole human brains (1 every other month). After fixation, processing 
  celloidin embedding, the whole brain
  will be serially cut at 100um thick. Each brain section will be 5 inches
  x 4.5 inches in size.
 
  I will given 250 of these whole brain sections to stain for tau
  IHC...that's 1500 whole brain sections/year!!!
  1) Does anyone have experience doing manual IHC staining of large
  free-floating brain sections?
  2) What type of staining tools, dishes or other essential equipment can
  anyone recommend?
  3) What's the most efficient way to stain 250 sections for batch IHC
  staining - such as transferring batch
  sections (maybe 5-10) from reagent to reagent?
  4) What type of batch apparatus to use?
 
  As for the antibody  ABC steps, I was thinking of placing each section
  inside a large glass cigar tube
  (yep, people use large glass tubes with fitted cap to store cigars), with
  5ml of antibody or ABC reagent  gently agitate on
  a shaker/rotator at room temp during the incubation. Does anyone have
  ideas on this?
 
  Please, any ideas, suggestions or recommendation anyone can provide will
  be most greatly appreciated.
 
  Best regards
  Maria Mejia
  UCSF
  Department of Neurology
  San Francisco, CA
 
 
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 http

Re: [Histonet] HELP! Need some old fashioned histology advice

[Jay Lundgren]

I can help with the old fashioned advice:


   - 1 scant teaspoon simple syrup
   - 2 dashes Angostura Bitters, plus more to taste
   - 1 half dollar–sized slice orange peel, including pith
   - 2 ounces good-quality rye or bourbon
   - 1 maraschino cherry

 As for the Histology, is there any reason you cannot mount the
sections onto glass slides?  When I was working at Genentech they were
cutting frozen sections through whole rabbits and mounting the sections on
(giant) glass slides.  I think that rolling the tissue up, inserting it,
and then removing it from a glass tube would destroy the tissue.

  Sincerely,

Jay A. Lundgren, M.S., HTL
(ASCP)

On Sat, Jan 3, 2015 at 8:01 PM, Maria Mejia mbmph...@gmail.com wrote:

 First, the very best of holidays to everyone.

 Now for the histology part.   Our lab's focus is on the early stages of
 Alzheimer's Disease in the Brainstem
 using celloidin processing  embedding for IHC staining.  This year, our
 lab will be receiving 6 post-mortem
 whole human brains (1 every other month).  After fixation, processing 
 celloidin embedding, the whole brain
 will be serially cut at 100um thick.  Each brain section will be 5 inches
 x 4.5 inches in size.

 I will given 250 of these whole brain sections to stain for tau
 IHC...that's 1500 whole brain sections/year!!!
 1) Does anyone have experience doing manual IHC staining of large
 free-floating brain sections?
 2) What type of staining tools, dishes or other essential equipment can
 anyone recommend?
 3) What's the most efficient way to stain 250 sections for batch IHC
 staining - such as transferring batch
 sections (maybe 5-10) from reagent to reagent?
 4) What type of batch apparatus to use?

 As for the antibody  ABC steps, I was thinking of placing each section
 inside a large glass cigar tube
 (yep, people use large glass tubes with fitted cap to store cigars), with
 5ml of antibody or ABC reagent  gently agitate on
 a shaker/rotator at room temp during the incubation.  Does anyone have
 ideas on this?

 Please, any ideas, suggestions or recommendation anyone can provide will
 be most greatly appreciated.

 Best regards
 Maria Mejia
 UCSF
 Department of Neurology
 San Francisco, CA


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RE: [Histonet] HELP! Need some old fashioned histology advice

[Patsy Ruegg]

I have done something similar to this but I used tissue that was fixed but not 
processed and embedded, this is called enblock labeling, I infiltrated the 
fixed tissue with the IHC reagents, in a vial/tube, the blocking reagents, then 
the antibody, then the detection reagents and DAB, then dehydrated the tissue.  
I used vials or tubes on a platform shaker and would infiltrate reagents for 
days, then after it was done I infiltrated and embedded the tissue in glycol 
methacrylate (GMA) so that I could section it, it actually worked.  The tissue 
was already IHc LABeled so all I did to the 5 micron sections after they were 
cut was a hematoxylin counterstain, this was mineralized bone so I had to 
embedd in something hard like GMA to section.

Will you remove the Celloidin before trying to do the IHC staining?  100 micron 
sections might be easy to float/handle using a glass pipette for transferring.  
Sounds like an interesting project, good luck and feel free to ask for advise 
and keep us posted on your progress.

Cheers,
Patsy

Patsy Ruegg, HT(ASCP)QIHC
Ruegg IHC Consulting
40864 E Arkansas Ave
Bennett, CO 80102
H 303-644-4538
C 720-281-5406
prueg...@hotmail.com



 Date: Sun, 4 Jan 2015 11:58:38 -0600
 From: jaylundg...@gmail.com
 To: mbmph...@gmail.com
 Subject: Re: [Histonet] HELP! Need some old fashioned histology advice
 CC: histonet@lists.utsouthwestern.edu; mary.me...@ucsf.edu
 
 I can help with the old fashioned advice:
 
 
- 1 scant teaspoon simple syrup
- 2 dashes Angostura Bitters, plus more to taste
- 1 half dollar–sized slice orange peel, including pith
- 2 ounces good-quality rye or bourbon
- 1 maraschino cherry
 
  As for the Histology, is there any reason you cannot mount the
 sections onto glass slides?  When I was working at Genentech they were
 cutting frozen sections through whole rabbits and mounting the sections on
 (giant) glass slides.  I think that rolling the tissue up, inserting it,
 and then removing it from a glass tube would destroy the tissue.
 
   Sincerely,
 
 Jay A. Lundgren, M.S., HTL
 (ASCP)
 
 On Sat, Jan 3, 2015 at 8:01 PM, Maria Mejia mbmph...@gmail.com wrote:
 
  First, the very best of holidays to everyone.
 
  Now for the histology part.   Our lab's focus is on the early stages of
  Alzheimer's Disease in the Brainstem
  using celloidin processing  embedding for IHC staining.  This year, our
  lab will be receiving 6 post-mortem
  whole human brains (1 every other month).  After fixation, processing 
  celloidin embedding, the whole brain
  will be serially cut at 100um thick.  Each brain section will be 5 inches
  x 4.5 inches in size.
 
  I will given 250 of these whole brain sections to stain for tau
  IHC...that's 1500 whole brain sections/year!!!
  1) Does anyone have experience doing manual IHC staining of large
  free-floating brain sections?
  2) What type of staining tools, dishes or other essential equipment can
  anyone recommend?
  3) What's the most efficient way to stain 250 sections for batch IHC
  staining - such as transferring batch
  sections (maybe 5-10) from reagent to reagent?
  4) What type of batch apparatus to use?
 
  As for the antibody  ABC steps, I was thinking of placing each section
  inside a large glass cigar tube
  (yep, people use large glass tubes with fitted cap to store cigars), with
  5ml of antibody or ABC reagent  gently agitate on
  a shaker/rotator at room temp during the incubation.  Does anyone have
  ideas on this?
 
  Please, any ideas, suggestions or recommendation anyone can provide will
  be most greatly appreciated.
 
  Best regards
  Maria Mejia
  UCSF
  Department of Neurology
  San Francisco, CA
 
 
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[Histonet] HELP! Need some old fashioned histology advice

[Maria Mejia]

First, the very best of holidays to everyone. 

Now for the histology part.   Our lab's focus is on the early stages of 
Alzheimer's Disease in the Brainstem 
using celloidin processing  embedding for IHC staining.  This year, our lab 
will be receiving 6 post-mortem
whole human brains (1 every other month).  After fixation, processing  
celloidin embedding, the whole brain 
will be serially cut at 100um thick.  Each brain section will be 5 inches x 4.5 
inches in size.  

I will given 250 of these whole brain sections to stain for tau IHC...that's 
1500 whole brain sections/year!!!
1) Does anyone have experience doing manual IHC staining of large free-floating 
brain sections?  
2) What type of staining tools, dishes or other essential equipment can anyone 
recommend?
3) What's the most efficient way to stain 250 sections for batch IHC staining - 
such as transferring batch 
sections (maybe 5-10) from reagent to reagent?  
4) What type of batch apparatus to use?

As for the antibody  ABC steps, I was thinking of placing each section inside 
a large glass cigar tube
(yep, people use large glass tubes with fitted cap to store cigars), with 5ml 
of antibody or ABC reagent  gently agitate on
a shaker/rotator at room temp during the incubation.  Does anyone have ideas on 
this?

Please, any ideas, suggestions or recommendation anyone can provide will be 
most greatly appreciated.

Best regards
Maria Mejia
UCSF 
Department of Neurology
San Francisco, CA


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[Histonet] 'Help'

[Marsha Price]

Sent from my iPhone

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[Histonet] Help with muscle

[Jennifer MacDonald]

Hello All,
I hope I am not asking a dumb question. As I know that everyone on the 
histonet is very knowledgeable I was hoping to get some suggestions 
regarding processing tissue for neuromuscular junction staining. We are a 
neuro research lab and want to quantify neuromuscular junction on treated 
and non-treated rat gastrocnemius. We need the whole muscle for 
quantifications. The first problem we encountered when attempting to do 
this on PFA perfused and post fixed whole muscle was negative staining 
with SV2 antibody (presynaptic) but nicely stained alpha bungarotoxin 
(post synaptic) end plates. After a few attempts with no success we 
decided to freeze the whole muscle in dry ice and isopentane. First off we 
are having issues with the middle of the muscle freezing completely even 
after leaving it in solution for more than 10 min. Secondly while we do 
get nice staining with SV2 and bungarotoxin in some of the endplates we 
don’t see colocalization in most of the NMJ’s. One possibility that I was 
thinking could be causing this is that when the muscle is being picked up 
on the slide (cryosections) it is not laying completely flat on the slide 
(we tend to have to focus back and forth quite a bit) so we see the 
SV2/bungarotoxin near each other but not overlapping. One other thing I 
thought might be occurring is that when the muscle is being frozen it is 
retracting causing a shift in the presynaptic/postsynaptic NMJ. What is 
the best way to process the tissue, fixation or freezing? Any suggestions 
are greatly appreciated.
Thanks,
Leslie
Leslie Garcia
Senior Histologist
Clive Svendsen Lab
Board of Governors Regenerative Medicine Institute Cedars-Sinai Medical 
Center
8700 Beverly Blvd.
AHSP 8405
Los Angeles, CA 90048
Phone - 310-248-8571
Web - http://www.cedars-sinai.edu/RMI
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[Histonet] HELP

[Hans B Snyder]

Hello All,

Does anyone have a protocol for the Anti-Neutrophil antibody [NIMP-R14]
(ab2557) for mouse tissue?  I have been trying to extinguish the background
staining but cannot find the right combination.
​ We are doing this by hand. ​
 The recommended dilution for this antibody is 1:50 with HIER.   See used
protocol below.

So far I have tried:
1. concentrations 1:25, 1:50, 1:75, 1:100  - 1:50 gives very high
background but good positive staining, 1:100 gives low background but not
much positive staining.

2. HIER - 3, 5, 10,  20, minutes  @ 96C, 20 minutes at 60C  all give the
same results - too much back ground

3. We have tried using the H2O2 before the primary and after, increasing
the time from 30 minutes to 45 minutes.

4. Tried incubating in the primary for shorter time (20 minutes) and longer
overnight at 4C.

5. Tried blocking using 3 different serums, first each one then
combinations and longer times.

6. Tried cutting the DAB concentration in 1/2 then 1/3.


We have not tried enzyme or acid epitope retrieval yet.



*Does anyone have a working protocol or suggestions on what to try?*


​Thank you in advance.​





Procedure:

Deparaffinize

1.  Heat slides to 60C for 10 minutes (oven).

2.  Dry slides at room temp 5 minutes.

3.  Xylene 5 minutes.

4.  Xylene 5 minutes.

5.  100% ethanol 5 minutes (dehydration).

6.  100% ethanol 2 minutes (dehydration).

7.  95% ethanol for 2 minutes (rehydration).

8.  70% ethanol for 2 minutes (rehydration).

9.   Run in DI water for 10 minutes.



Antigen Retrieval

1.  Submerge in 0.01M Citrate Buffer (pH 6.0) at 96C for 5 minutes.

2.  Cold running water 5 minutes.



Staining

1.  Wash in PBS for 1 min.

2.  Permeabilize with 0.1% Triton X / 2.5% horse serum + 2.5% fetal
bovine serum for 2 hours.

3.  Incubate in primary antibody 30-45 minutes at in 2.5% horse serum
in PBS.

4.  Wash 3x in PBS/tween for 5 minutes each.

5.  Fresh 3.0% hydrogen peroxide (H2O2) 2x for 20 minutes each.

6.  Wash 3x PBS 2 minutes each.

7.  Incubate with anti-Rat Ig impress solution (according to vector’s
instructions) 45 minutes.

8.  Wash 3x in PBS for 5 minutes each.

9.  Prepare Impact DAB substrate (see insert).

10.  Using a light microscope, incubate sections with 50-100ul, until
desired darkness occurs (5-30 seconds).

11.  Stop DAB reaction using deionized water.

12.  Wash in DH2O for 5 minutes

13.  Counterstain in Harris hematoxylin for 30 seconds.

14.  Wash in DH2O for 5 minutes.

15.  Dehydrate 2x in 95% alcohol for 30 seconds each.

16.  Dehydrate 3x in 100% alcohol for 2 minutes each.

17.  Clear 2x in xylene 5 minutes each.

18.  Mount coverslips using Leica mounting media.


Hans B Snyder
Histologistics
60 Prescott Street
Worcester, MA 01605
508-308-7800
h...@histologistics.com
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RE: [Histonet] HELP

[Elizabeth Chlipala]

Hans

We use the antibody from Serotec, it works quite nicely.  MCA711, proteinase K 
digestion,  rabbit anti-rat secondary and then Envision Rabbit (polymer).  We 
use this antibody at a 1:1200 dilution.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Hans B Snyder
Sent: Thursday, November 06, 2014 10:31 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] HELP

Hello All,

Does anyone have a protocol for the Anti-Neutrophil antibody [NIMP-R14]
(ab2557) for mouse tissue?  I have been trying to extinguish the background 
staining but cannot find the right combination.
​ We are doing this by hand. ​
 The recommended dilution for this antibody is 1:50 with HIER.   See used
protocol below.

So far I have tried:
1. concentrations 1:25, 1:50, 1:75, 1:100  - 1:50 gives very high background 
but good positive staining, 1:100 gives low background but not much positive 
staining.

2. HIER - 3, 5, 10,  20, minutes  @ 96C, 20 minutes at 60C  all give the same 
results - too much back ground

3. We have tried using the H2O2 before the primary and after, increasing the 
time from 30 minutes to 45 minutes.

4. Tried incubating in the primary for shorter time (20 minutes) and longer 
overnight at 4C.

5. Tried blocking using 3 different serums, first each one then combinations 
and longer times.

6. Tried cutting the DAB concentration in 1/2 then 1/3.


We have not tried enzyme or acid epitope retrieval yet.



*Does anyone have a working protocol or suggestions on what to try?*


​Thank you in advance.​





Procedure:

Deparaffinize

1.  Heat slides to 60C for 10 minutes (oven).

2.  Dry slides at room temp 5 minutes.

3.  Xylene 5 minutes.

4.  Xylene 5 minutes.

5.  100% ethanol 5 minutes (dehydration).

6.  100% ethanol 2 minutes (dehydration).

7.  95% ethanol for 2 minutes (rehydration).

8.  70% ethanol for 2 minutes (rehydration).

9.   Run in DI water for 10 minutes.



Antigen Retrieval

1.  Submerge in 0.01M Citrate Buffer (pH 6.0) at 96C for 5 minutes.

2.  Cold running water 5 minutes.



Staining

1.  Wash in PBS for 1 min.

2.  Permeabilize with 0.1% Triton X / 2.5% horse serum + 2.5% fetal
bovine serum for 2 hours.

3.  Incubate in primary antibody 30-45 minutes at in 2.5% horse serum
in PBS.

4.  Wash 3x in PBS/tween for 5 minutes each.

5.  Fresh 3.0% hydrogen peroxide (H2O2) 2x for 20 minutes each.

6.  Wash 3x PBS 2 minutes each.

7.  Incubate with anti-Rat Ig impress solution (according to vector’s
instructions) 45 minutes.

8.  Wash 3x in PBS for 5 minutes each.

9.  Prepare Impact DAB substrate (see insert).

10.  Using a light microscope, incubate sections with 50-100ul, until desired 
darkness occurs (5-30 seconds).

11.  Stop DAB reaction using deionized water.

12.  Wash in DH2O for 5 minutes

13.  Counterstain in Harris hematoxylin for 30 seconds.

14.  Wash in DH2O for 5 minutes.

15.  Dehydrate 2x in 95% alcohol for 30 seconds each.

16.  Dehydrate 3x in 100% alcohol for 2 minutes each.

17.  Clear 2x in xylene 5 minutes each.

18.  Mount coverslips using Leica mounting media.


Hans B Snyder
Histologistics
60 Prescott Street
Worcester, MA 01605
508-308-7800
h...@histologistics.com
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RE: [Histonet] HELP

[Connolly, Brett M]

We also use the MCA711 antibody (at 1.0 ug/mL, 1 hr.), but with HIER in citrate 
buffer using a Biocare Decloaker pressure cooker and Biocare's rat-on mouse HRP 
polymer detection. We  get very nice staining with no background. I was never 
too impressed with ImPRESS.

Here are the basics :
- Deparaffinize and hydrate to dH20
- Perform HIER, cool for 20 min then wash in H20
- 3.0% H2O2 - 20 min
- Serum free block (Biocare Sniper) 30 min
- Incubate with MCA771 1 hr.
- Incubate with Biocare rat-on-Mouse kit (per instructions)
- DAB - 5 min.
 Washes are with PBS/0.1% Tween

Brett


Brett M. Connolly, Ph.D.
Principle Scientist, Imaging Dept.
Merck  Co., Inc.
PO Box 4, WP-44K
West Point, PA 19486
brett_conno...@merck.com
T- 215-652-2501
F- 215-993-6803




-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Elizabeth 
Chlipala
Sent: Thursday, November 06, 2014 12:41 PM
To: Hans B Snyder; histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] HELP

Hans

We use the antibody from Serotec, it works quite nicely.  MCA711, proteinase K 
digestion,  rabbit anti-rat secondary and then Envision Rabbit (polymer).  We 
use this antibody at a 1:1200 dilution.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Hans B Snyder
Sent: Thursday, November 06, 2014 10:31 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] HELP

Hello All,

Does anyone have a protocol for the Anti-Neutrophil antibody [NIMP-R14]
(ab2557) for mouse tissue?  I have been trying to extinguish the background 
staining but cannot find the right combination.
​ We are doing this by hand. ​
 The recommended dilution for this antibody is 1:50 with HIER.   See used
protocol below.

So far I have tried:
1. concentrations 1:25, 1:50, 1:75, 1:100  - 1:50 gives very high background 
but good positive staining, 1:100 gives low background but not much positive 
staining.

2. HIER - 3, 5, 10,  20, minutes  @ 96C, 20 minutes at 60C  all give the same 
results - too much back ground

3. We have tried using the H2O2 before the primary and after, increasing the 
time from 30 minutes to 45 minutes.

4. Tried incubating in the primary for shorter time (20 minutes) and longer 
overnight at 4C.

5. Tried blocking using 3 different serums, first each one then combinations 
and longer times.

6. Tried cutting the DAB concentration in 1/2 then 1/3.


We have not tried enzyme or acid epitope retrieval yet.



*Does anyone have a working protocol or suggestions on what to try?*


​Thank you in advance.​





Procedure:

Deparaffinize

1.  Heat slides to 60C for 10 minutes (oven).

2.  Dry slides at room temp 5 minutes.

3.  Xylene 5 minutes.

4.  Xylene 5 minutes.

5.  100% ethanol 5 minutes (dehydration).

6.  100% ethanol 2 minutes (dehydration).

7.  95% ethanol for 2 minutes (rehydration).

8.  70% ethanol for 2 minutes (rehydration).

9.   Run in DI water for 10 minutes.



Antigen Retrieval

1.  Submerge in 0.01M Citrate Buffer (pH 6.0) at 96C for 5 minutes.

2.  Cold running water 5 minutes.



Staining

1.  Wash in PBS for 1 min.

2.  Permeabilize with 0.1% Triton X / 2.5% horse serum + 2.5% fetal
bovine serum for 2 hours.

3.  Incubate in primary antibody 30-45 minutes at in 2.5% horse serum
in PBS.

4.  Wash 3x in PBS/tween for 5 minutes each.

5.  Fresh 3.0% hydrogen peroxide (H2O2) 2x for 20 minutes each.

6.  Wash 3x PBS 2 minutes each.

7.  Incubate with anti-Rat Ig impress solution (according to vector’s
instructions) 45 minutes.

8.  Wash 3x in PBS for 5 minutes each.

9.  Prepare Impact DAB substrate (see insert).

10.  Using a light microscope, incubate sections with 50-100ul, until desired 
darkness occurs (5-30 seconds).

11.  Stop DAB reaction using deionized water.

12.  Wash in DH2O for 5 minutes

13.  Counterstain in Harris hematoxylin for 30 seconds.

14.  Wash in DH2O for 5 minutes.

15.  Dehydrate 2x in 95% alcohol for 30 seconds each.

16.  Dehydrate 3x in 100% alcohol for 2 minutes each.

17.  Clear 2x in xylene 5 minutes each.

18.  Mount coverslips using Leica mounting media.


Hans B Snyder
Histologistics
60 Prescott Street
Worcester, MA 01605
508-308-7800
h...@histologistics.com
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[Histonet] Help! Need someone to repair an IEC CTD Cryostat

[Mercedes Gallagher]

Hi,

I'm having extreme difficulty finding someone who can work on the old IEC
CTDs in Southern California.  The issue isn't with the microtome, it's with
the compressor/refrigeration.  I've had a few people tell me they deal with
these machines, only to find out after many hours of labor that they don't
have a clue.

Does anyone have any contacts?

Thanks!
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[Histonet] Help Please - Need a Leica Rep to contact me

[Wolfe, Christina]

I am in Indiana - I need a Leica sales rep to contact me please or if someone 
can provide me with a regional sales rep name - that would be awesome!
Thanks!
Kristie

Ph 812-307-2093
christina.wo...@bms.com


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[Histonet] HELP- Cryosectioning FAT!

[Balasubbramanian, Dakshnapriya]

Hi all,

I've been having problems with cryosectioning fatty tissue for a long time now. 
The section leaves a hole in the middle. I know this is probably a much 
discussed topic, but I've tried a lot of strategies with no luck. The tissue is 
of mouse origin and has micro-lesions buried inside fat. So the fat needs to be 
cut in order to get to the lesion. I started with a temperature of -17C and 
tried upto -25C and also at -50C; didn't work at any of these temperatures. I 
tried rubbing the block, blade and anti-roll plate with dry ice-again,no luck. 

I rapid-freeze the tissue with OCT in LN2 and then store the block at -80C. 
Could I try sectioning the block directly from -80C? (never done that). 

Any other suggestions on how to tackle this?

Any advice is much appreciated!!

Thanks,
Dakshna

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Re: [Histonet] HELP- Cryosectioning FAT!

[Balasubbramanian, Dakshnapriya]

Thanks Sandra! My next step was to try that.

Dakshna

- Original Message -
From: Sandra E. Esparza sespa...@seton.org
To: Dakshnapriya Balasubbramanian dakshnapr...@neo.tamu.edu
Sent: Tuesday, May 20, 2014 10:34:25 AM
Subject: RE: [Histonet] HELP- Cryosectioning FAT!

You might want to dry Liquid Nitrogen on the face of the block before you cut 
it.  

Sandra

Sandra Esparza HT (ASCP), QIHC
Histotechnologist Mohs
Austin Dermatologic Surgery Center
512-324-7468  x84027
sespa...@seton.org


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of 
Balasubbramanian, Dakshnapriya
Sent: Tuesday, May 20, 2014 10:23 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] HELP- Cryosectioning FAT!

Hi all,

I've been having problems with cryosectioning fatty tissue for a long time now. 
The section leaves a hole in the middle. I know this is probably a much 
discussed topic, but I've tried a lot of strategies with no luck. The tissue is 
of mouse origin and has micro-lesions buried inside fat. So the fat needs to be 
cut in order to get to the lesion. I started with a temperature of -17C and 
tried upto -25C and also at -50C; didn't work at any of these temperatures. I 
tried rubbing the block, blade and anti-roll plate with dry ice-again,no luck. 

I rapid-freeze the tissue with OCT in LN2 and then store the block at -80C. 
Could I try sectioning the block directly from -80C? (never done that). 

Any other suggestions on how to tackle this?

Any advice is much appreciated!!

Thanks,
Dakshna

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Re: [Histonet] HELP- Cryosectioning FAT!

[Balasubbramanian, Dakshnapriya]

Hi Laurie,

No, the tissue hasn't been fixed before freezing. My prof doesn't prefer the 
method, but if nothing else works, we might give it a try. 

Any suggested protocols for fixation?

Thanks!
Dakshna

- Original Message -
From: Laurie J King king.lau...@marshfieldclinic.org
To: Dakshnapriya Balasubbramanian dakshnapr...@neo.tamu.edu
Sent: Tuesday, May 20, 2014 10:37:35 AM
Subject: RE: [Histonet] HELP- Cryosectioning FAT!

Dakshna,

Has this tissue been fixed before freezing by any chance?

laurie

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of 
Balasubbramanian, Dakshnapriya
Sent: Tuesday, May 20, 2014 10:23 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] HELP- Cryosectioning FAT!

Hi all,

I've been having problems with cryosectioning fatty tissue for a long time now. 
The section leaves a hole in the middle. I know this is probably a much 
discussed topic, but I've tried a lot of strategies with no luck. The tissue is 
of mouse origin and has micro-lesions buried inside fat. So the fat needs to be 
cut in order to get to the lesion. I started with a temperature of -17C and 
tried upto -25C and also at -50C; didn't work at any of these temperatures. I 
tried rubbing the block, blade and anti-roll plate with dry ice-again,no luck. 

I rapid-freeze the tissue with OCT in LN2 and then store the block at -80C. 
Could I try sectioning the block directly from -80C? (never done that). 

Any other suggestions on how to tackle this?

Any advice is much appreciated!!

Thanks,
Dakshna

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Re: [Histonet] Help for identifying the blue stained structure

[Marvin Hanna]

Hi Rui,

You uploaded a tif image and a number of browsers don't support tif 
images. Jpeg, gif and png images are the best image formats to use 
because they are universally supported. I converted your image to a jpeg 
image and posted it at:


http://histosearch.com/imageupload/help-for-identifying-the-blue-stained-structure-jpeg/

Best Regards,

Marvin Hanna


On 05/17/2014 06:39 PM, Rui TAHARA wrote:


Hello,
I have an embryonic sample that
decalcified, paraffin embedded, and stained with Mallory Trichrome (Aniline
Blue, Orange G, Acid Fuchsin). I will upload the image in the Histonet Images. 
This
is a cranial region where the bone is being resorbed, so I expected to see the
bone (dark blue in trabecular), and red blood, and adjacent white spaces that
is being resorbed. Instead, there is a very uniform, granular structure stained
with blue without any nuclei at the resorbed regions. This structure looks like
almost crystal or some kind of secretion leakage from the ossifying bone. I
need a help to identify this blue stained things. I don't think this is osteoid,
because at later stage embryos, there is no bone at this region.

If you have some suggestions for other
stain to identify this blue thing, or help identifying this, I really
appreciate it.

  


Thank you in advance,

  


Rui




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RE: [Histonet] Help for identifying the blue stained structure

[Rui TAHARA]

Hi, 

Thank you so much. 
I was wondering why the image has not been seen on the site, and thought its 
been processed. 

Thank you for uploading the image. 

Rui 

Date: Sun, 18 May 2014 17:47:21 -0400
From: mha...@histosearch.com
To: ru...@hotmail.com; histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Help for identifying the blue stained structure


  

  
  
Hi Rui,



You uploaded a tif image and a number of browsers don't support tif
images. Jpeg, gif and png images are the best image formats to use
because they are universally supported. I converted your image to a
jpeg image and posted it at:




http://histosearch.com/imageupload/help-for-identifying-the-blue-stained-structure-jpeg/



Best Regards,



Marvin Hanna





On 05/17/2014 06:39 PM, Rui TAHARA
  wrote:



  Hello, 
I have an embryonic sample that
decalcified, paraffin embedded, and stained with Mallory Trichrome (Aniline
Blue, Orange G, Acid Fuchsin). I will upload the image in the Histonet Images. 
This
is a cranial region where the bone is being resorbed, so I expected to see the
bone (dark blue in trabecular), and red blood, and adjacent white spaces that
is being resorbed. Instead, there is a very uniform, granular structure stained
with blue without any nuclei at the resorbed regions. This structure looks like
almost crystal or some kind of secretion leakage from the ossifying bone. I
need a help to identify this blue stained things. I don’t think this is osteoid,
because at later stage embryos, there is no bone at this region. 

If you have some suggestions for other
stain to identify this blue thing, or help identifying this, I really
appreciate it. 

 

Thank you in advance, 

 

Rui 

  
  

  
  

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[Histonet] Help for identifying the blue stained structure

[Rui TAHARA]

Hello, 
I have an embryonic sample that
decalcified, paraffin embedded, and stained with Mallory Trichrome (Aniline
Blue, Orange G, Acid Fuchsin). I will upload the image in the Histonet Images. 
This
is a cranial region where the bone is being resorbed, so I expected to see the
bone (dark blue in trabecular), and red blood, and adjacent white spaces that
is being resorbed. Instead, there is a very uniform, granular structure stained
with blue without any nuclei at the resorbed regions. This structure looks like
almost crystal or some kind of secretion leakage from the ossifying bone. I
need a help to identify this blue stained things. I don’t think this is osteoid,
because at later stage embryos, there is no bone at this region. 

If you have some suggestions for other
stain to identify this blue thing, or help identifying this, I really
appreciate it. 

 

Thank you in advance, 

 

Rui 

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[Histonet] Help kappa and lambda IHC on bone marrow bxs

[drmoses111]

Our lab has started using Immunocal decal solution on our bone marrows. Most of 
our antibodies have improved except kappa and lambda. We do not do ISH. Kappa 
and lambda staining in the tonsil controls is good, The bone marrows are now 
very overstrained. We use DAKO polyclonals  at 1:10,000 with protease1 on the 
Ventana Ultra. Does anyone have a procedure? 
- Original Message -

  
  
From: histonet-requ...@lists.utsouthwestern.edu 
To: histonet@lists.utsouthwestern.edu 
Subject: Histonet Digest, Vol 124, Issue 24 

Send Histonet mailing list submissions to 
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When replying, please edit your Subject line so it is more specific 
than Re: Contents of Histonet digest... 


Today's Topics: 

   1. specimen marking ink (Davis, Cassie) 
   2. Leica Reichert Jung Cryocut 1800 (King, Laurie J) 
   3. RE: specimen marking ink (wanda.sm...@hcahealthcare.com) 
   4. IHC on paraffin embedded skin tissue! (Jennifer Leigh) 
   5. Re: RE: specimen marking ink (David Kemler) 
   6. Re: RE: specimen marking ink (Bryan Llewellyn) 
   7. Re: IHC on paraffin embedded skin tissue! (C.D.G.) 


-- 

Message: 1 
Date: Fri, 21 Mar 2014 13:03:35 -0400 
From: Davis, Cassie cda...@che-east.org 
Subject: [Histonet] specimen marking ink 
To: histonet@lists.utsouthwestern.edu 
histonet@lists.utsouthwestern.edu 
Message-ID: 
be7af02009c3a74f9b8344e70f3655cbfb9...@chexcms01.one.ads.che.org 
Content-Type: text/plain; charset=iso-8859-1 

Hi Histo World, as I was cutting to day I was thinking why don't we see if we 
could get specimen marking ink directly from a tattoo vendor? When I first 
started in histo I was told the ink we use was actually tattoo ink. As we know 
as soon as somebody labels something as a medical supply the price is 
increased. Just a cost saving thought, I mentioned it to my immediate 
supervisor but she think it would be a liability issue. I thought we could 
test/validate it on skin tissue left over from a mastectomy or extremity. Any 
thoughts? 

Cassandra Davis 
cda...@che-east.org 
302-575-8095 



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-- 

Message: 2 
Date: Fri, 21 Mar 2014 17:45:48 + 
From: King, Laurie J king.lau...@marshfieldclinic.org 
Subject: [Histonet] Leica Reichert Jung Cryocut 1800 
To: 'histonet@lists.utsouthwestern.edu' 
(histonet@lists.utsouthwestern.edu) 
histonet@lists.utsouthwestern.edu 
Message-ID: 
7578207839f50248a7a6cd33517295ea4f13b...@mcl-exmb03.mfldclin.org 
Content-Type: text/plain; charset=us-ascii 

Hello all, 

Looking for a manual for a Leica Reichert Jung Cryocut 1800. 

Laurie 

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Message: 3 
Date: Fri, 21 Mar 2014 14:37:59 -0500 
From: wanda.sm...@hcahealthcare.com 
Subject: [Histonet] RE: specimen marking ink 
To: cda...@che-east.org, histonet@lists.utsouthwestern.edu 
Message-ID: 

9e2d36ce2d7cba4a94d9b22e8328a3ba27efddf...@nadcwpmsgcms03.hca.corpad.net 
 
Content-Type: text/plain; charset=iso-8859-1 

Back in the 70's (when I was six, but still a histotech!!!) my Pathologist had 
gotten tattoo pigment powder in two colors and that's what we used to mark 
tissue.  Don't know where it came from and don't know where it went!!! 
Happy Friday and Happy Weekend to everyone!!! 
Wanda 

WANDA G. SMITH, HTL(ASCP)HT 
Pathology Supervisor 
TRIDENT MEDICAL CENTER 
9330 Medical Plaza Drive 
Charleston, SC� 29406 
843-847-4586 
843-847-4296 fax 

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[Histonet] Help on get PDF file paper from J. Histotechnology

[Dorothy Hu]

Hi Histonetters,

Would you please let me know why I can not get PDF file from J
Histotechnology any more?

I renewed my membership after new year and changed my password since i
don't remember  my old one. I can get in Maney Online for only Abstract,
but not whole article.

The paper I tried to get is:
Parallel experience of two different lab with initiator perkadox 16 for
polymerization of MMA.
Vol 17, No 4, pp.343-348.

Thanks in advance.

Dorothy
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Re: [Histonet] Help on get PDF file paper from J. Histotechnology

[Dorothy Hu]

Thanks very much Jean. I will call NSH office. Dorothy


On Fri, Jan 17, 2014 at 2:53 PM, Mitchell Jean A jmitch...@uwhealth.orgwrote:

 Dorothy:  I know that there have been some issues with Maney Online
 lately.  I suggest your contact the NSH office and they should be able to
 assist you.  I had the same problem last week and it was handled very
 quickly.


 Jean Mitchell, BS HT (ASCP)
 University of Wisconsin Hospital  Clinics
 Neuromuscular Laboratory Manager
 600 Highland Avenue
 Madison, WI  53792-5132


 -Original Message-
 From: histonet-boun...@lists.utsouthwestern.edu [mailto:
 histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Dorothy Hu
 Sent: Friday, January 17, 2014 8:57 AM
 To: histonet@lists.utsouthwestern.edu
 Subject: [Histonet] Help on get PDF file paper from J. Histotechnology

 Hi Histonetters,

 Would you please let me know why I can not get PDF file from J
 Histotechnology any more?

 I renewed my membership after new year and changed my password since i
 don't remember  my old one. I can get in Maney Online for only Abstract,
 but not whole article.

 The paper I tried to get is:
 Parallel experience of two different lab with initiator perkadox 16 for
 polymerization of MMA.
 Vol 17, No 4, pp.343-348.

 Thanks in advance.

 Dorothy
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Re: [Histonet] Help

[Emily Sours]

FACEPALM.JPG

this is not even worth an actual jpg.
sigh.

By bitching and bitching and bitching, they could exhaust the drama of
their own horror stories. Grow bored. Only then could they accept a new
story for their lives. Move forward.

-Chuck Palahniuk, Haunted


On Wed, Sep 11, 2013 at 7:51 PM, Jay Lundgren jaylundg...@gmail.com wrote:

 lol


 On Wed, Sep 11, 2013 at 5:56 PM, Bain,Virginia veb...@mdanderson.org
 wrote:

  Googling 'unsubscribe histonet' points me to this link:
  http://lists.utsouthwestern.edu/mailman/listinfo/histonet
 
  If you scroll to the bottom of the page you will find a box where you can
  type your e-mail address to unsubscribe or edit options.  Have you tried
  that?
 
  Cheers.
 
  --
  Virginia Bain
  Postdoctoral Fellow
  Richie Lab
  512-237-6443
 
 
 
 
 
  On 9/11/13 5:38 PM, nancy mcvay njmcvay...@gmail.com wrote:
 
  Get me off your mailing list.
  I  have tried and keep getting these things.
  Over and over you repeat the same questions and answers ,   over and
 over,
  over  and over,  over and over,  over and over.
  
  Do  you get the message?
  Do you get the message?
  Do you get the message?
  Get me off your mailing list
  Get me off your mailing list
  
  
  Today  I received four different email  tirades from you.
  
  
  Please get me off your mailing list.
  
  I am to the point of reporting it as spam.
  
  njmcvay...@gmail.com
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[Histonet] Help

[nancy mcvay]

Get me off your mailing list.
I  have tried and keep getting these things.
Over and over you repeat the same questions and answers ,   over and over,
over  and over,  over and over,  over and over.

Do  you get the message?
Do you get the message?
Do you get the message?
Get me off your mailing list
Get me off your mailing list


Today  I received four different email  tirades from you.


Please get me off your mailing list.

I am to the point of reporting it as spam.

njmcvay...@gmail.com
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Re: [Histonet] Help

[Bain,Virginia]

Googling 'unsubscribe histonet' points me to this link:
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

If you scroll to the bottom of the page you will find a box where you can
type your e-mail address to unsubscribe or edit options.  Have you tried
that?

Cheers.

-- 
Virginia Bain
Postdoctoral Fellow
Richie Lab
512-237-6443





On 9/11/13 5:38 PM, nancy mcvay njmcvay...@gmail.com wrote:

Get me off your mailing list.
I  have tried and keep getting these things.
Over and over you repeat the same questions and answers ,   over and over,
over  and over,  over and over,  over and over.

Do  you get the message?
Do you get the message?
Do you get the message?
Get me off your mailing list
Get me off your mailing list


Today  I received four different email  tirades from you.


Please get me off your mailing list.

I am to the point of reporting it as spam.

njmcvay...@gmail.com
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Re: [Histonet] Help

[Jay Lundgren]

lol


On Wed, Sep 11, 2013 at 5:56 PM, Bain,Virginia veb...@mdanderson.orgwrote:

 Googling 'unsubscribe histonet' points me to this link:
 http://lists.utsouthwestern.edu/mailman/listinfo/histonet

 If you scroll to the bottom of the page you will find a box where you can
 type your e-mail address to unsubscribe or edit options.  Have you tried
 that?

 Cheers.

 --
 Virginia Bain
 Postdoctoral Fellow
 Richie Lab
 512-237-6443





 On 9/11/13 5:38 PM, nancy mcvay njmcvay...@gmail.com wrote:

 Get me off your mailing list.
 I  have tried and keep getting these things.
 Over and over you repeat the same questions and answers ,   over and over,
 over  and over,  over and over,  over and over.
 
 Do  you get the message?
 Do you get the message?
 Do you get the message?
 Get me off your mailing list
 Get me off your mailing list
 
 
 Today  I received four different email  tirades from you.
 
 
 Please get me off your mailing list.
 
 I am to the point of reporting it as spam.
 
 njmcvay...@gmail.com
 ___
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[Histonet] Help for Northern CA HT program

[Jennifer MacDonald]

Merritt College has started a HT program and is in need of clinical sites 
for their students to get hands-on experience.  If you interested in 
being a mentor for the HT students please contact Gisele Giorgi at 
profgio...@peralta.edu
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[Histonet] HELP please!

[Jaclyn Pitts]

Hey everyone.
I Need some help with my processor. It is a thermo scientific stp 120. I
came in today and when I went to puch the button to shut the alarm off, I
was shocked. The machine shocked me and then the little green display
screen went blank. I managed to get my blocks off the machine and I think
that I could still run programs but without the display working how am I to
know what program it is on!! What can I do? The manual doesnt say anything
about what to do about this.

-- 

*Jaclyn Pitts, HT(ASCP)CM*

218-454-3520

Dermatology Professionals, PA

15167 Edgewood Dr. Suite 200

Baxter, MN 56425
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[Histonet] Help!

[Richard Cartun]

I need to speak with someone who has experience with doing (and interpretating) 
in situ hybridization for EBER.  Please contact me directly.  Thank you.

Richard

Richard W. Cartun, MS, PhD
Director, Histology  Immunopathology
Director, Biospecimen Collection Programs
Assistant Director, Anatomic Pathology
Hartford Hospital
80 Seymour Street
Hartford, CT  06102
(860) 545-1596
(860) 545-2204 Fax




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RE: [Histonet] Help with CPT code 88363 for archived tissue retrieval

[Weems, Joyce K.]

Yes, it is for review of material for molecular testing and can be added at any 
time as long as it is not ordered at the time the case is in process. If it is 
more than 30 days after the patient has been discharged, it is considered 
archived (according to Medicare) and we register it for a new acct number and 
bill it alone. Otherwise it is a late bill on current visit.

We do this regardless if the patient is Medicare or not - just for consistency. 
I am not aware of any problems.

Joyce Weems
Pathology Manager
678-843-7376 Phone
678-843-7831 Fax
joyce.we...@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-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Natalie Nagy
Sent: Thursday, January 10, 2013 10:36 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Help with CPT code 88363 for archived tissue retrieval

Hi everyone,
   I just have a question about CPT code 88363, first can it be 
used for pulling blocks for Oncotype DX testing, also is there a time limit on 
when this code can be used? Does it have to be within a year, a month, etc...of 
when the patient account went active?

Thanks for all the help,

Natalie J. Nagy (HT)ASCP
Histology Supervisor
Holyoke Medical Center


CONFIDENTIALITY NOTICE: This email communication and any attachments may 
contain confidential and privileged information for the use of the designated 
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[Histonet] Help with CPT code 88363 for archived tissue retrieval

[Natalie Nagy]

Hi everyone,
   I just have a question about CPT code 88363, first can it be 
used for pulling blocks for Oncotype DX testing, also is there a time limit on 
when this code can be used? Does it have to be within a year, a month, etc...of 
when the patient account went active?
 
Thanks for all the help,
 
Natalie J. Nagy (HT)ASCP
Histology Supervisor
Holyoke Medical Center


CONFIDENTIALITY NOTICE: This email communication and any attachments may 
contain confidential and privileged information for the use of the designated 
recipients named above. If you are not the intended recipient, you are hereby 
notified that you have received this communication in error and that any 
review, disclosure, dissemination, distribution or copying of it or its 
contents is prohibited. If you have received this communication in error, 
please reply to the sender immediately and destroy all copies of this 
communication and any attachments. For further information regarding Holyoke 
Medical Center's privacy policy, Please visit our Internet web site at 
http://www.holyokehealth.com
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Re: [Histonet] Help with CPT code 88363 for archived tissue retrieval

[Mark Tarango]

It can't be used to just pull blocks.  The slides have to be reviewed and
the best block chosen by a pathologist.  If there is only one block then
the pathologist needs to look at the slides and determine if there is
enough tissue for molecular testing.  It's a professional charge.

Use it on re-accessioned cases for which molecular testing is requested and
the best block needs to be chosen.

Yes we use it when sending out for Oncotype DX if the case was signed out
over 30 days before.

Mark

On Thu, Jan 10, 2013 at 7:36 AM, Natalie Nagy 
nagy_nata...@holyokehealth.com wrote:

 Hi everyone,
I just have a question about CPT code 88363, first can
 it be used for pulling blocks for Oncotype DX testing, also is there a time
 limit on when this code can be used? Does it have to be within a year, a
 month, etc...of when the patient account went active?

 Thanks for all the help,

 Natalie J. Nagy (HT)ASCP
 Histology Supervisor
 Holyoke Medical Center


 CONFIDENTIALITY NOTICE: This email communication and any attachments may
 contain confidential and privileged information for the use of the
 designated recipients named above. If you are not the intended recipient,
 you are hereby notified that you have received this communication in error
 and that any review, disclosure, dissemination, distribution or copying of
 it or its contents is prohibited. If you have received this communication
 in error, please reply to the sender immediately and destroy all copies of
 this communication and any attachments. For further information regarding
 Holyoke Medical Center's privacy policy, Please visit our Internet web site
 at http://www.holyokehealth.com
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RE: [Histonet] Help with CPT code 88363 for archived tissue retrieval

[Weems, Joyce K.]

And I should explain the reason I most know this.. our pathologists were denied 
because the tech charge hadn't been entered yet. So now I make sure the tech 
charge is entered before sending to the pathologists billing folks.

Joyce Weems
Pathology Manager
678-843-7376 Phone
678-843-7831 Fax
joyce.we...@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-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Mark Tarango
Sent: Thursday, January 10, 2013 2:01 PM
To: Natalie Nagy
Cc: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Help with CPT code 88363 for archived tissue retrieval

It can't be used to just pull blocks.  The slides have to be reviewed and the 
best block chosen by a pathologist.  If there is only one block then the 
pathologist needs to look at the slides and determine if there is enough tissue 
for molecular testing.  It's a professional charge.

Use it on re-accessioned cases for which molecular testing is requested and the 
best block needs to be chosen.

Yes we use it when sending out for Oncotype DX if the case was signed out over 
30 days before.

Mark

On Thu, Jan 10, 2013 at 7:36 AM, Natalie Nagy  nagy_nata...@holyokehealth.com 
wrote:

 Hi everyone,
I just have a question about CPT code 88363, first
 can it be used for pulling blocks for Oncotype DX testing, also is
 there a time limit on when this code can be used? Does it have to be
 within a year, a month, etc...of when the patient account went active?

 Thanks for all the help,

 Natalie J. Nagy (HT)ASCP
 Histology Supervisor
 Holyoke Medical Center


 CONFIDENTIALITY NOTICE: This email communication and any attachments
 may contain confidential and privileged information for the use of the
 designated recipients named above. If you are not the intended
 recipient, you are hereby notified that you have received this
 communication in error and that any review, disclosure, dissemination,
 distribution or copying of it or its contents is prohibited. If you
 have received this communication in error, please reply to the sender
 immediately and destroy all copies of this communication and any
 attachments. For further information regarding Holyoke Medical
 Center's privacy policy, Please visit our Internet web site at
 http://www.holyokehealth.com
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RE: [Histonet] Help with CPT code 88363 for archived tissue retrieval

[Weems, Joyce K.]

It is also a technical charge, as well.

Joyce Weems
Pathology Manager
678-843-7376 Phone
678-843-7831 Fax
joyce.we...@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-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Mark Tarango
Sent: Thursday, January 10, 2013 2:01 PM
To: Natalie Nagy
Cc: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Help with CPT code 88363 for archived tissue retrieval

It can't be used to just pull blocks.  The slides have to be reviewed and the 
best block chosen by a pathologist.  If there is only one block then the 
pathologist needs to look at the slides and determine if there is enough tissue 
for molecular testing.  It's a professional charge.

Use it on re-accessioned cases for which molecular testing is requested and the 
best block needs to be chosen.

Yes we use it when sending out for Oncotype DX if the case was signed out over 
30 days before.

Mark

On Thu, Jan 10, 2013 at 7:36 AM, Natalie Nagy  nagy_nata...@holyokehealth.com 
wrote:

 Hi everyone,
I just have a question about CPT code 88363, first
 can it be used for pulling blocks for Oncotype DX testing, also is
 there a time limit on when this code can be used? Does it have to be
 within a year, a month, etc...of when the patient account went active?

 Thanks for all the help,

 Natalie J. Nagy (HT)ASCP
 Histology Supervisor
 Holyoke Medical Center


 CONFIDENTIALITY NOTICE: This email communication and any attachments
 may contain confidential and privileged information for the use of the
 designated recipients named above. If you are not the intended
 recipient, you are hereby notified that you have received this
 communication in error and that any review, disclosure, dissemination,
 distribution or copying of it or its contents is prohibited. If you
 have received this communication in error, please reply to the sender
 immediately and destroy all copies of this communication and any
 attachments. For further information regarding Holyoke Medical
 Center's privacy policy, Please visit our Internet web site at
 http://www.holyokehealth.com
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RE: [Histonet] Help with CPT code 88363 for archived tissue retrieval

[Mike Pence]

So are you putting the charge thru twice or is the charge for the 88363
divided out into tech and prof.?

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Weems,
Joyce K.
Sent: Thursday, January 10, 2013 1:06 PM
To: 'Mark Tarango'; Natalie Nagy
Cc: histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] Help with CPT code 88363 for archived tissue
retrieval


And I should explain the reason I most know this.. our pathologists were
denied because the tech charge hadn't been entered yet. So now I make
sure the tech charge is entered before sending to the pathologists
billing folks.

Joyce Weems
Pathology Manager
678-843-7376 Phone
678-843-7831 Fax
joyce.we...@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-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Mark
Tarango
Sent: Thursday, January 10, 2013 2:01 PM
To: Natalie Nagy
Cc: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Help with CPT code 88363 for archived tissue
retrieval

It can't be used to just pull blocks.  The slides have to be reviewed
and the best block chosen by a pathologist.  If there is only one block
then the pathologist needs to look at the slides and determine if there
is enough tissue for molecular testing.  It's a professional charge.

Use it on re-accessioned cases for which molecular testing is requested
and the best block needs to be chosen.

Yes we use it when sending out for Oncotype DX if the case was signed
out over 30 days before.

Mark

On Thu, Jan 10, 2013 at 7:36 AM, Natalie Nagy 
nagy_nata...@holyokehealth.com wrote:

 Hi everyone,
I just have a question about CPT code 88363, first 
 can it be used for pulling blocks for Oncotype DX testing, also is 
 there a time limit on when this code can be used? Does it have to be 
 within a year, a month, etc...of when the patient account went active?

 Thanks for all the help,

 Natalie J. Nagy (HT)ASCP
 Histology Supervisor
 Holyoke Medical Center


 CONFIDENTIALITY NOTICE: This email communication and any attachments 
 may contain confidential and privileged information for the use of the

 designated recipients named above. If you are not the intended 
 recipient, you are hereby notified that you have received this 
 communication in error and that any review, disclosure, dissemination,

 distribution or copying of it or its contents is prohibited. If you 
 have received this communication in error, please reply to the sender 
 immediately and destroy all copies of this communication and any 
 attachments. For further information regarding Holyoke Medical 
 Center's privacy policy, Please visit our Internet web site at 
 http://www.holyokehealth.com 
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RE: [Histonet] Help with CPT code 88363 for archived tissue retrieval

[Weems, Joyce K.]

Divided - but can be billed globally if that is how you bill. The professional 
uses the 26 modifier. 

Joyce Weems
Pathology Manager
678-843-7376 Phone
678-843-7831 Fax
joyce.we...@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: Mike Pence [mailto:mpe...@grhs.net] 
Sent: Thursday, January 10, 2013 2:28 PM
To: Weems, Joyce K.; Mark Tarango; Natalie Nagy
Cc: histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] Help with CPT code 88363 for archived tissue retrieval

So are you putting the charge thru twice or is the charge for the 88363 divided 
out into tech and prof.?

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Weems, Joyce K.
Sent: Thursday, January 10, 2013 1:06 PM
To: 'Mark Tarango'; Natalie Nagy
Cc: histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] Help with CPT code 88363 for archived tissue retrieval


And I should explain the reason I most know this.. our pathologists were denied 
because the tech charge hadn't been entered yet. So now I make sure the tech 
charge is entered before sending to the pathologists billing folks.

Joyce Weems
Pathology Manager
678-843-7376 Phone
678-843-7831 Fax
joyce.we...@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-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Mark Tarango
Sent: Thursday, January 10, 2013 2:01 PM
To: Natalie Nagy
Cc: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Help with CPT code 88363 for archived tissue retrieval

It can't be used to just pull blocks.  The slides have to be reviewed and the 
best block chosen by a pathologist.  If there is only one block then the 
pathologist needs to look at the slides and determine if there is enough tissue 
for molecular testing.  It's a professional charge.

Use it on re-accessioned cases for which molecular testing is requested and the 
best block needs to be chosen.

Yes we use it when sending out for Oncotype DX if the case was signed out over 
30 days before.

Mark

On Thu, Jan 10, 2013 at 7:36 AM, Natalie Nagy  nagy_nata...@holyokehealth.com 
wrote:

 Hi everyone,
I just have a question about CPT code 88363, first 
 can it be used for pulling blocks for Oncotype DX testing, also is 
 there a time limit on when this code can be used? Does it have to be 
 within a year, a month, etc...of when the patient account went active?

 Thanks for all the help,

 Natalie J. Nagy (HT)ASCP
 Histology Supervisor
 Holyoke Medical Center


 CONFIDENTIALITY NOTICE: This email communication and any attachments 
 may contain confidential and privileged information for the use of the

 designated recipients named above. If you are not the intended 
 recipient, you are hereby notified that you have received this 
 communication in error and that any review, disclosure, dissemination,

 distribution or copying of it or its contents is prohibited. If you 
 have received this communication in error, please reply to the sender 
 immediately and destroy all copies of this communication and any 
 attachments. For further information regarding Holyoke Medical 
 Center's privacy policy, Please visit our Internet web site at 
 http://www.holyokehealth.com 
 ___
 Histonet mailing list
 Histonet@lists.utsouthwestern.edu
 http://lists.utsouthwestern.edu/mailman/listinfo/histonet

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(including any attachments

RE: [Histonet] Help with CPT code 88363 for archived tissue retrieval

[Richard Cartun]

I'm not 100% sure, but I don't think this CPT code has a Technical component.

Richard

Richard W. Cartun, MS, PhD
Director, Histology  Immunopathology
Director, Biospecimen Collection Programs
Assistant Director, Anatomic Pathology
Hartford Hospital
80 Seymour Street
Hartford, CT  06102
(860) 545-1596
(860) 545-2204 Fax


 Mike Pence mpe...@grhs.net 1/10/2013 2:27 PM 
So are you putting the charge thru twice or is the charge for the 88363
divided out into tech and prof.?

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Weems,
Joyce K.
Sent: Thursday, January 10, 2013 1:06 PM
To: 'Mark Tarango'; Natalie Nagy
Cc: histonet@lists.utsouthwestern.edu 
Subject: RE: [Histonet] Help with CPT code 88363 for archived tissue
retrieval


And I should explain the reason I most know this.. our pathologists were
denied because the tech charge hadn't been entered yet. So now I make
sure the tech charge is entered before sending to the pathologists
billing folks.

Joyce Weems
Pathology Manager
678-843-7376 Phone
678-843-7831 Fax
joyce.we...@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-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Mark
Tarango
Sent: Thursday, January 10, 2013 2:01 PM
To: Natalie Nagy
Cc: histonet@lists.utsouthwestern.edu 
Subject: Re: [Histonet] Help with CPT code 88363 for archived tissue
retrieval

It can't be used to just pull blocks.  The slides have to be reviewed
and the best block chosen by a pathologist.  If there is only one block
then the pathologist needs to look at the slides and determine if there
is enough tissue for molecular testing.  It's a professional charge.

Use it on re-accessioned cases for which molecular testing is requested
and the best block needs to be chosen.

Yes we use it when sending out for Oncotype DX if the case was signed
out over 30 days before.

Mark

On Thu, Jan 10, 2013 at 7:36 AM, Natalie Nagy 
nagy_nata...@holyokehealth.com wrote:

 Hi everyone,
I just have a question about CPT code 88363, first 
 can it be used for pulling blocks for Oncotype DX testing, also is 
 there a time limit on when this code can be used? Does it have to be 
 within a year, a month, etc...of when the patient account went active?

 Thanks for all the help,

 Natalie J. Nagy (HT)ASCP
 Histology Supervisor
 Holyoke Medical Center


 CONFIDENTIALITY NOTICE: This email communication and any attachments 
 may contain confidential and privileged information for the use of the

 designated recipients named above. If you are not the intended 
 recipient, you are hereby notified that you have received this 
 communication in error and that any review, disclosure, dissemination,

 distribution or copying of it or its contents is prohibited. If you 
 have received this communication in error, please reply to the sender 
 immediately and destroy all copies of this communication and any 
 attachments. For further information regarding Holyoke Medical 
 Center's privacy policy, Please visit our Internet web site at 
 http://www.holyokehealth.com 
 ___
 Histonet mailing list
 Histonet@lists.utsouthwestern.edu 
 http://lists.utsouthwestern.edu/mailman/listinfo/histonet 

___
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If you have received this message in error, please contact
the sender by reply e-mail message and destroy all copies of the
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Histonet

RE: [Histonet] Help with CPT code 88363 for archived tissue retrieval

[Bell, Lynne]

I agree, Dr. Cartun.  I believe that the CPT Coding book specifically says that 
it is only a professional charge.

Lynne Bell, HT (ASCP)
Histology Team Leader
Central Vermont Medical Center
130 Fisher Road
Berlin, VT  05641
802-371-4923
-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Richard Cartun
Sent: Thursday, January 10, 2013 2:47 PM
To: Joyce K. Weems; Mark Tarango; Mike Pence; Natalie Nagy
Cc: histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] Help with CPT code 88363 for archived tissue retrieval

I'm not 100% sure, but I don't think this CPT code has a Technical component.

Richard

Richard W. Cartun, MS, PhD
Director, Histology  Immunopathology
Director, Biospecimen Collection Programs
Assistant Director, Anatomic Pathology
Hartford Hospital
80 Seymour Street
Hartford, CT  06102
(860) 545-1596
(860) 545-2204 Fax


 Mike Pence mpe...@grhs.net 1/10/2013 2:27 PM 
So are you putting the charge thru twice or is the charge for the 88363
divided out into tech and prof.?

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Weems,
Joyce K.
Sent: Thursday, January 10, 2013 1:06 PM
To: 'Mark Tarango'; Natalie Nagy
Cc: histonet@lists.utsouthwestern.edu 
Subject: RE: [Histonet] Help with CPT code 88363 for archived tissue
retrieval


And I should explain the reason I most know this.. our pathologists were
denied because the tech charge hadn't been entered yet. So now I make
sure the tech charge is entered before sending to the pathologists
billing folks.

Joyce Weems
Pathology Manager
678-843-7376 Phone
678-843-7831 Fax
joyce.we...@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-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Mark
Tarango
Sent: Thursday, January 10, 2013 2:01 PM
To: Natalie Nagy
Cc: histonet@lists.utsouthwestern.edu 
Subject: Re: [Histonet] Help with CPT code 88363 for archived tissue
retrieval

It can't be used to just pull blocks.  The slides have to be reviewed
and the best block chosen by a pathologist.  If there is only one block
then the pathologist needs to look at the slides and determine if there
is enough tissue for molecular testing.  It's a professional charge.

Use it on re-accessioned cases for which molecular testing is requested
and the best block needs to be chosen.

Yes we use it when sending out for Oncotype DX if the case was signed
out over 30 days before.

Mark

On Thu, Jan 10, 2013 at 7:36 AM, Natalie Nagy 
nagy_nata...@holyokehealth.com wrote:

 Hi everyone,
I just have a question about CPT code 88363, first 
 can it be used for pulling blocks for Oncotype DX testing, also is 
 there a time limit on when this code can be used? Does it have to be 
 within a year, a month, etc...of when the patient account went active?

 Thanks for all the help,

 Natalie J. Nagy (HT)ASCP
 Histology Supervisor
 Holyoke Medical Center


 CONFIDENTIALITY NOTICE: This email communication and any attachments 
 may contain confidential and privileged information for the use of the

 designated recipients named above. If you are not the intended 
 recipient, you are hereby notified that you have received this 
 communication in error and that any review, disclosure, dissemination,

 distribution or copying of it or its contents is prohibited. If you 
 have received this communication in error, please reply to the sender 
 immediately and destroy all copies of this communication and any 
 attachments. For further information regarding Holyoke Medical 
 Center's privacy policy, Please visit our Internet web site at 
 http://www.holyokehealth.com 
 ___
 Histonet mailing list
 Histonet@lists.utsouthwestern.edu 
 http://lists.utsouthwestern.edu/mailman/listinfo/histonet 

___
Histonet mailing list
Histonet@lists.utsouthwestern.edu 
http://lists.utsouthwestern.edu/mailman/listinfo/histonet 



This e-mail message (including any attachments) is for the sole use of
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recipient, you are hereby notified that any dissemination, distribution
or copying of this message (including any attachments

RE: [Histonet] Help with CPT code 88363 for archived tissue retrieval

[Weems, Joyce K.]

Maybe so, but I am getting reimbursed for tech only. 

Joyce Weems
Pathology Manager
678-843-7376 Phone
678-843-7831 Fax
joyce.we...@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: Richard Cartun [mailto:rcar...@harthosp.org] 
Sent: Thursday, January 10, 2013 2:47 PM
To: Weems, Joyce K.; Mark Tarango; Mike Pence; Natalie Nagy
Cc: histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] Help with CPT code 88363 for archived tissue retrieval

I'm not 100% sure, but I don't think this CPT code has a Technical component.

Richard

Richard W. Cartun, MS, PhD
Director, Histology  Immunopathology
Director, Biospecimen Collection Programs Assistant Director, Anatomic 
Pathology Hartford Hospital
80 Seymour Street
Hartford, CT  06102
(860) 545-1596
(860) 545-2204 Fax


 Mike Pence mpe...@grhs.net 1/10/2013 2:27 PM 
So are you putting the charge thru twice or is the charge for the 88363 divided 
out into tech and prof.?

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Weems, Joyce K.
Sent: Thursday, January 10, 2013 1:06 PM
To: 'Mark Tarango'; Natalie Nagy
Cc: histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] Help with CPT code 88363 for archived tissue retrieval


And I should explain the reason I most know this.. our pathologists were denied 
because the tech charge hadn't been entered yet. So now I make sure the tech 
charge is entered before sending to the pathologists billing folks.

Joyce Weems
Pathology Manager
678-843-7376 Phone
678-843-7831 Fax
joyce.we...@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-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Mark Tarango
Sent: Thursday, January 10, 2013 2:01 PM
To: Natalie Nagy
Cc: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Help with CPT code 88363 for archived tissue retrieval

It can't be used to just pull blocks.  The slides have to be reviewed and the 
best block chosen by a pathologist.  If there is only one block then the 
pathologist needs to look at the slides and determine if there is enough tissue 
for molecular testing.  It's a professional charge.

Use it on re-accessioned cases for which molecular testing is requested and the 
best block needs to be chosen.

Yes we use it when sending out for Oncotype DX if the case was signed out over 
30 days before.

Mark

On Thu, Jan 10, 2013 at 7:36 AM, Natalie Nagy  nagy_nata...@holyokehealth.com 
wrote:

 Hi everyone,
I just have a question about CPT code 88363, first 
 can it be used for pulling blocks for Oncotype DX testing, also is 
 there a time limit on when this code can be used? Does it have to be 
 within a year, a month, etc...of when the patient account went active?

 Thanks for all the help,

 Natalie J. Nagy (HT)ASCP
 Histology Supervisor
 Holyoke Medical Center


 CONFIDENTIALITY NOTICE: This email communication and any attachments 
 may contain confidential and privileged information for the use of the

 designated recipients named above. If you are not the intended 
 recipient, you are hereby notified that you have received this 
 communication in error and that any review, disclosure, dissemination,

 distribution or copying of it or its contents is prohibited. If you 
 have received this communication in error, please reply to the sender 
 immediately and destroy all copies of this communication and any 
 attachments. For further information regarding Holyoke Medical 
 Center's privacy policy, Please visit our Internet web site at 
 http://www.holyokehealth.com 
 ___
 Histonet mailing list
 Histonet@lists.utsouthwestern.edu
 http://lists.utsouthwestern.edu/mailman/listinfo/histonet

___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

RE: [Histonet] Help with CPT code 88363 for archived tissue retrieval

[Weems, Joyce K.]

Same here - and we are being reimbursed. We don't charge much. Medicare new 
rate is $12.71 - but every billable test helps when that is what our 
productivity is based on! 

Joyce Weems
Pathology Manager
678-843-7376 Phone
678-843-7831 Fax
joyce.we...@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: Victor A. Tobias [mailto:vtob...@uw.edu] 
Sent: Thursday, January 10, 2013 5:47 PM
To: 'Bell, Lynne'; 'Richard Cartun'; Weems, Joyce K.; 'Mark Tarango'; 'Mike 
Pence'; 'Natalie Nagy'
Cc: 'histonet@lists.utsouthwestern.edu'
Subject: RE: [Histonet] Help with CPT code 88363 for archived tissue retrieval

We have been billing a tech and pro fee since 2011. Whether we are getting 
reimbursed for both is another question.

Victor

Victor Tobias HT(ASCP)
Clinical Applications Analyst
Harborview Medical Center
Dept of Pathology Room NJB244
Seattle, WA 98104
vtob...@u.washington.edu
206-744-2735
206-744-8240 Fax
=
Privileged, confidential or patient identifiable information may be contained 
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addressed to you in error, do not read, disclose, reproduce, distribute, 
disseminate or otherwise use this transmission. Instead, please notify the 
sender by reply e-mail, and then destroy all copies of the message and any 
attachments.

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Bell, Lynne
Sent: Thursday, January 10, 2013 11:50 AM
To: 'Richard Cartun'; Joyce K. Weems; Mark Tarango; Mike Pence; Natalie Nagy
Cc: histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] Help with CPT code 88363 for archived tissue retrieval

I agree, Dr. Cartun.  I believe that the CPT Coding book specifically says that 
it is only a professional charge.

Lynne Bell, HT (ASCP)
Histology Team Leader
Central Vermont Medical Center
130 Fisher Road
Berlin, VT  05641
802-371-4923
-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Richard Cartun
Sent: Thursday, January 10, 2013 2:47 PM
To: Joyce K. Weems; Mark Tarango; Mike Pence; Natalie Nagy
Cc: histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] Help with CPT code 88363 for archived tissue retrieval

I'm not 100% sure, but I don't think this CPT code has a Technical component.

Richard

Richard W. Cartun, MS, PhD
Director, Histology  Immunopathology
Director, Biospecimen Collection Programs Assistant Director, Anatomic 
Pathology Hartford Hospital
80 Seymour Street
Hartford, CT  06102
(860) 545-1596
(860) 545-2204 Fax


 Mike Pence mpe...@grhs.net 1/10/2013 2:27 PM 
So are you putting the charge thru twice or is the charge for the 88363 divided 
out into tech and prof.?

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Weems, Joyce K.
Sent: Thursday, January 10, 2013 1:06 PM
To: 'Mark Tarango'; Natalie Nagy
Cc: histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] Help with CPT code 88363 for archived tissue retrieval


And I should explain the reason I most know this.. our pathologists were denied 
because the tech charge hadn't been entered yet. So now I make sure the tech 
charge is entered before sending to the pathologists billing folks.

Joyce Weems
Pathology Manager
678-843-7376 Phone
678-843-7831 Fax
joyce.we...@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-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Mark Tarango
Sent: Thursday, January 10, 2013 2:01 PM
To: Natalie Nagy
Cc: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Help with CPT code 88363 for archived tissue retrieval

It can't be used to just pull blocks.  The slides have to be reviewed

[Histonet] Help

[Ly Nguyen]

I want to be remove from the list.  I've tried the unsubscribe link be low
several time but it seem like it doesn't work because I'm still receiving
daily email

On Friday, December 28, 2012, wrote:

 Send Histonet mailing list submissions to
 histonet@lists.utsouthwestern.edu

 To subscribe or unsubscribe via the World Wide Web, visit
 http://lists.utsouthwestern.edu/mailman/listinfo/histonet
 or, via email, send a message with subject or body 'help' to
 histonet-requ...@lists.utsouthwestern.edu

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 histonet-ow...@lists.utsouthwestern.edu

 When replying, please edit your Subject line so it is more specific
 than Re: Contents of Histonet digest...


 Today's Topics:

1. Microtome maintenance frequency (Allen Keeping D.)
2. Melanin Bleaching (Koehnlein, Melissa)
3. RE: Microtome maintenance frequency (Morken, Timothy)
4. Re: Microtome maintenance frequency (Rene J Buesa)
5. Tissue Microarray Question (Jim Jones)
6. In permeabilization,  can we use Tween-20 instead of
   Triton-100? ()


 --

 Message: 1
 Date: Thu, 27 Dec 2012 11:40:23 -0700
 From: Allen Keeping D. allen.keep...@albertahealthservices.ca
 Subject: [Histonet] Microtome maintenance frequency
 To: 'histonet@lists.utsouthwestern.edu'
 histonet@lists.utsouthwestern.edu
 Message-ID:
 
 1dc4211314f7d9458b66a28f947f60b901236e95d...@exmbx4c.crha.bewell.ca
 Content-Type: text/plain; charset=us-ascii

 I have a question regarding scheduled maintenance of rotary microtomes. I
 run a simulation lab for students of lab technology. Part of their
 histology rotation involves learning how to produce acceptable sections on
 the microtome. I currently have 3 Leica rotary (manual) microtomes, which
 are used by students a total of 18-20 days per year (for educational
 purposes, not to produce diagnostic materials). In addition, I may use a
 microtome for a couple weeks total each year to produce control materials
 and to troubleshoot staining issues.

 Currently, the microtomes are serviced by a Leica-educated professional
 annually for preventative maintenance. As these microtomes are only used
 infrequently, is this level of maintenance necessary (or recommended)? I am
 contemplating switching to a bi-annual schedule as a cost-saving measure.

 Does anyone have any advice or experience regarding maintaining microtomes
 in a low-volume setting?

 Cheers,

 Allen



   
 This message and any attached documents are only for the use of the
 intended recipient(s), are confidential and may contain privileged
 information. Any unauthorized review, use, retransmission, or other
 disclosure is strictly prohibited. If you have received this message in
 error, please notify the sender immediately, and then delete the original
 message. Thank you.


 --

 Message: 2
 Date: Thu, 27 Dec 2012 19:04:29 +
 From: Koehnlein, Melissa melissa.koehnl...@pds.usask.ca
 Subject: [Histonet] Melanin Bleaching
 To: histonet@lists.utsouthwestern.edu
 histonet@lists.utsouthwestern.edu
 Message-ID:
 87bf46bacb9b124ead75f536e82a9bede0b...@campusmb5.usask.ca
 Content-Type: text/plain; charset=us-ascii

 Melanin can be distinguished from DAB by counterstaining with Azure B,
 which gives a dark green color to the melanin.  We've used the procedure
 below with success:

 Immunoperoxidase technique modified by counterstain with azure B as a
 diagnostic aid in evaluating heavily pigmented melanocytic neoplasms:

 http://onlinelibrary.wiley.com/doi/10./j.1600-0560.1991.tb01381.x/abstract

 Melissa Koehnlein
 Lab Technician
 Prairie Diagnostic Services Inc.
 52 Campus Drive, Saskatoon, SK S7N 5B4
 Phone (306) 966-7223
 Fax (306) 966-2488
 Email mailto:melissa.koehnl...@pds.usask.ca

 
 From: histonet-boun...@lists.utsouthwestern.edu [
 histonet-boun...@lists.utsouthwestern.edu] on behalf of
 histonet-requ...@lists.utsouthwestern.edu [
 histonet-requ...@lists.utsouthwestern.edu]
 Sent: Thursday, December 27, 2012 12:03 PM
 To: histonet@lists.utsouthwestern.edu
 Subject: Histonet Digest, Vol 109, Issue 29

 Send Histonet mailing list submissions to
 histonet@lists.utsouthwestern.edu

 To subscribe or unsubscribe via the World Wide Web, visit
 http://lists.utsouthwestern.edu/mailman/listinfo/histonet
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 than Re: Contents of Histonet digest...


 Today's Topics:

1. Melanin Bleaching (Janci Wellborn)
2. Re: Melanin Bleaching (Rene J Buesa)


 

Re: [Histonet] Help

[Marvin Hanna]

Hi Ly,

When you click on the Unsubscribe button, an email is sent to you to 
confirm you want to unsubscribe. It has a link to click to confirm you 
want to unsubscribe. Is the confirmation email getting lost in your junk 
folder? You won't be unsubscribed until you click the link in the 
confirmation email. The confirmation email should have a subject like, 
confirm 1a5b and a return address of 
histonet-requ...@list.utsouthwestern.edu. Hope this helps.


Best Regards,

Marvin Hanna

On 12/28/2012 2:04 PM, Ly Nguyen wrote:

I want to be remove from the list.  I've tried the unsubscribe link be low
several time but it seem like it doesn't work because I'm still receiving
daily email





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RE: [Histonet] Help

[Bartlett, Jeanine (CDC/OID/NCEZID)]

For some reason I just received 3 emails from Histonet asking me to confirm if 
I want to be unsubscribedI did not request to be unsubscribed.

Did anyone else receive emails like this?

Jeanine H. Bartlett
Centers for Disease Control and Prevention
Infectious Diseases Pathology Branch
404-639-3590
jeanine.bartl...@cdc.hhs.gov

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Marvin Hanna
Sent: Friday, December 28, 2012 2:28 PM
To: histonet@lists.utsouthwestern.edu; ln0...@gmail.com
Subject: Re: [Histonet] Help

Hi Ly,

When you click on the Unsubscribe button, an email is sent to you to confirm 
you want to unsubscribe. It has a link to click to confirm you want to 
unsubscribe. Is the confirmation email getting lost in your junk folder? You 
won't be unsubscribed until you click the link in the confirmation email. The 
confirmation email should have a subject like, confirm 1a5b and a return 
address of histonet-requ...@list.utsouthwestern.edu. Hope this helps.

Best Regards,

Marvin Hanna

On 12/28/2012 2:04 PM, Ly Nguyen wrote:
 I want to be remove from the list.  I've tried the unsubscribe link be 
 low several time but it seem like it doesn't work because I'm still 
 receiving daily email




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Re: [Histonet] Help

[Marvin Hanna]

Hi Jeanine,

Hmmm... The only way that should happen is if someone put your email 
address in and clicked unsubscribe 3 times. That's the purpose of the 
confirmation email so that no one else can unsubscribe (or subscribe) 
you. So, anyone out there trying to unsubscribe Jeanine, please stop. 
The computer server is smarter than you. And we like Jeanine. :-)


Marvin

On 12/28/2012 2:33 PM, Bartlett, Jeanine (CDC/OID/NCEZID) wrote:

For some reason I just received 3 emails from Histonet asking me to confirm if 
I want to be unsubscribedI did not request to be unsubscribed.

Did anyone else receive emails like this?

Jeanine H. Bartlett
Centers for Disease Control and Prevention
Infectious Diseases Pathology Branch
404-639-3590
jeanine.bartl...@cdc.hhs.gov

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Marvin Hanna
Sent: Friday, December 28, 2012 2:28 PM
To: histonet@lists.utsouthwestern.edu; ln0...@gmail.com
Subject: Re: [Histonet] Help

Hi Ly,

When you click on the Unsubscribe button, an email is sent to you to confirm you want to 
unsubscribe. It has a link to click to confirm you want to unsubscribe. Is the confirmation email getting 
lost in your junk folder? You won't be unsubscribed until you click the link in the confirmation email. The 
confirmation email should have a subject like, confirm 1a5b and a return address of 
histonet-requ...@list.utsouthwestern.edu. Hope this helps.

Best Regards,

Marvin Hanna

On 12/28/2012 2:04 PM, Ly Nguyen wrote:

I want to be remove from the list.  I've tried the unsubscribe link be
low several time but it seem like it doesn't work because I'm still
receiving daily email




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[Histonet] help with myelin staining in poorly osmicated tissue

[Lange, Mollie]

Is there an alternative to toluidine blue stain for plastic embedded tissue?
I have some valuable epoxy resin embedded rat spinal cord blocks that were not 
well osmicated.  Toluidine Blue staining of the semi-thin sections is uneven 
and pale in the center of the sections.  Is there another stain I could try 
that is not dependent on good osmium penetration?  I fear that alcohol 
dehydration and propylene oxide clearing may have demyelinated the poorly 
osmicated portions of the block.  In that case, no stain will work.  But I'm 
not ready to give up yet.

Thanks for any and all advice.  I've been out of the lab for a while.

Mollie Lange
Project Manager
International Center for Spinal Cord Injury
Huo W. Moser Research Institute at Kennedy Krieger
707 N. Broadway
Baltimore, MD 21205
443-923-9241 phone
443-923-9245 fax
la...@kennedykrieger.orgmailto:la...@kennedykrieger.org

Disclaimer:
The materials in this e-mail are private and may contain Protected Health 
Information. Please note that e-mail is not necessarily confidential or secure. 
Your use of e-mail constitutes your acknowledgment of these confidentiality and 
security limitations. If you are not the intended recipient, be advised that 
any unauthorized use, disclosure, copying, distribution, or the taking of any 
action in reliance on the contents of this information is strictly prohibited. 
If you have received this e-mail in error, please immediately notify the sender 
via telephone or return e-mail.
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RE: [Histonet] Help! Liver mistakenly processed in paraffin (had tobe in OCT instead)!

[Patsy Ruegg]

Yea if you were looking for fat that will be lost with paraffin processing,
so the oil red o for fat will not work.  I would not see any point in trying
to depara and then snap freeze, but if I did want to do that since the
tissue is formalin fixed I would depara, fix some more in formalin and then
infiltrate the tissue in 30% sucrose overnight before snap freezing, if you
do not do that fix tissue will not section well frozen.

Patsy Ruegg, HT(ASCP)QIHC
Ruegg IHC Consulting, LLC
40864 Arkansas Ave
Bennett, CO 80102
Phone: 303-644-4538
Fax: 720-859-4110
pru...@ihctech.net

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Jennifer
MacDonald
Sent: Tuesday, November 13, 2012 10:15 AM
To: z o n k e d
Cc: histonet@lists.utsouthwestern.edu;
histonet-boun...@lists.utsouthwestern.edu
Subject: Re: [Histonet] Help! Liver mistakenly processed in paraffin (had
tobe in OCT instead)!

It depends on what you are using the oil red o for.  Lipofuscin and ceroid 
can be demonstrated with an oil red o stain after processing.
Jennifer MacDonald




From:   z o n k e d zon...@gmail.com
To: histonet@lists.utsouthwestern.edu 
histonet@lists.utsouthwestern.edu
Date:   11/13/2012 08:53 AM
Subject:[Histonet] Help! Liver mistakenly processed in paraffin 
(had to be  in OCT instead)!
Sent by:histonet-boun...@lists.utsouthwestern.edu



Hello Histonetters,

First time writer, long time reader. I'm a newbie tech in academia and I
was given a simple task which I think I pretty much screwed up.

I should have embedded half of a mouse liver in paraffin for microtome
sectioning while the other half should have been embedded in OCT for
cryosectioning (for oil red o). I made the mistake last night of placing
both liver halves into the tissue processor. The liver I intended for OCT
embedding is now hard as wax. Is there any way to deparaffinize processed
organs and may I embed them in OCT for proper cryosectioning? I imagine
that the liver would get dehydrated, I would get crappy sections, and Oil
Red O won't work.

Any suggestions are welcome.

Thank you so much,

Zoe W.


-- 
It costs nothing to say something kind. Even less to shut up altogether.

--Nathan Fillion
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[Histonet] Help! Liver mistakenly processed in paraffin (had to be in OCT instead)!

[z o n k e d]

Hello Histonetters,

First time writer, long time reader. I'm a newbie tech in academia and I
was given a simple task which I think I pretty much screwed up.

I should have embedded half of a mouse liver in paraffin for microtome
sectioning while the other half should have been embedded in OCT for
cryosectioning (for oil red o). I made the mistake last night of placing
both liver halves into the tissue processor. The liver I intended for OCT
embedding is now hard as wax. Is there any way to deparaffinize processed
organs and may I embed them in OCT for proper cryosectioning? I imagine
that the liver would get dehydrated, I would get crappy sections, and Oil
Red O won't work.

Any suggestions are welcome.

Thank you so much,

Zoe W.


-- 
It costs nothing to say something kind. Even less to shut up altogether.

--Nathan Fillion
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Re: [Histonet] Help! Liver mistakenly processed in paraffin (had to be in OCT instead)!

[Will Chappell]

Nope, sorry. All your fat is dissolved. 

Sent from my iPhone

On Nov 13, 2012, at 8:52 AM, z o n k e d zon...@gmail.com wrote:

 Hello Histonetters,
 
 First time writer, long time reader. I'm a newbie tech in academia and I
 was given a simple task which I think I pretty much screwed up.
 
 I should have embedded half of a mouse liver in paraffin for microtome
 sectioning while the other half should have been embedded in OCT for
 cryosectioning (for oil red o). I made the mistake last night of placing
 both liver halves into the tissue processor. The liver I intended for OCT
 embedding is now hard as wax. Is there any way to deparaffinize processed
 organs and may I embed them in OCT for proper cryosectioning? I imagine
 that the liver would get dehydrated, I would get crappy sections, and Oil
 Red O won't work.
 
 Any suggestions are welcome.
 
 Thank you so much,
 
 Zoe W.
 
 
 -- 
 It costs nothing to say something kind. Even less to shut up altogether.
 
--Nathan Fillion
 ___
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 Histonet@lists.utsouthwestern.edu
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Re: [Histonet] Help! Liver mistakenly processed in paraffin (had to be in OCT instead)!

[Rena Fail]

Oil Red O is a stain for fat, Alcohol dissolves fat Rehydrating  won't
help. You  can't replace the fat.
Rena Fail

On Tue, Nov 13, 2012 at 11:52 AM, z o n k e d zon...@gmail.com wrote:

 Hello Histonetters,

 First time writer, long time reader. I'm a newbie tech in academia and I
 was given a simple task which I think I pretty much screwed up.

 I should have embedded half of a mouse liver in paraffin for microtome
 sectioning while the other half should have been embedded in OCT for
 cryosectioning (for oil red o). I made the mistake last night of placing
 both liver halves into the tissue processor. The liver I intended for OCT
 embedding is now hard as wax. Is there any way to deparaffinize processed
 organs and may I embed them in OCT for proper cryosectioning? I imagine
 that the liver would get dehydrated, I would get crappy sections, and Oil
 Red O won't work.

 Any suggestions are welcome.

 Thank you so much,

 Zoe W.


 --
 It costs nothing to say something kind. Even less to shut up altogether.

 --Nathan Fillion
 ___
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 Histonet@lists.utsouthwestern.edu
 http://lists.utsouthwestern.edu/mailman/listinfo/histonet

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Re: [Histonet] Help! Liver mistakenly processed in paraffin (had to be in OCT instead)!

[Jennifer MacDonald]

It depends on what you are using the oil red o for.  Lipofuscin and ceroid 
can be demonstrated with an oil red o stain after processing.
Jennifer MacDonald




From:   z o n k e d zon...@gmail.com
To: histonet@lists.utsouthwestern.edu 
histonet@lists.utsouthwestern.edu
Date:   11/13/2012 08:53 AM
Subject:[Histonet] Help! Liver mistakenly processed in paraffin 
(had to be  in OCT instead)!
Sent by:histonet-boun...@lists.utsouthwestern.edu



Hello Histonetters,

First time writer, long time reader. I'm a newbie tech in academia and I
was given a simple task which I think I pretty much screwed up.

I should have embedded half of a mouse liver in paraffin for microtome
sectioning while the other half should have been embedded in OCT for
cryosectioning (for oil red o). I made the mistake last night of placing
both liver halves into the tissue processor. The liver I intended for OCT
embedding is now hard as wax. Is there any way to deparaffinize processed
organs and may I embed them in OCT for proper cryosectioning? I imagine
that the liver would get dehydrated, I would get crappy sections, and Oil
Red O won't work.

Any suggestions are welcome.

Thank you so much,

Zoe W.


-- 
It costs nothing to say something kind. Even less to shut up altogether.

--Nathan Fillion
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Re: [Histonet] Help! Liver mistakenly processed in paraffin (had to be in OCT instead)!

[Rene J Buesa]

You really screwed it up!
When you placed both pieces of liver in the processor both were subjected to 
the effect of ethanol and probably xylene and both reagents extracted the liver 
fat and no matter what you try to do now, there will be not enough fat in the 
pieces as to even try the ORO stain.
Try to get another piece. Anything you will try will not render good 
cryosections and no fat staining.
René J.



From: z o n k e d zon...@gmail.com
To: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu 
Sent: Tuesday, November 13, 2012 11:52 AM
Subject: [Histonet] Help! Liver mistakenly processed in paraffin (had to be in 
OCT instead)!

Hello Histonetters,

First time writer, long time reader. I'm a newbie tech in academia and I
was given a simple task which I think I pretty much screwed up.

I should have embedded half of a mouse liver in paraffin for microtome
sectioning while the other half should have been embedded in OCT for
cryosectioning (for oil red o). I made the mistake last night of placing
both liver halves into the tissue processor. The liver I intended for OCT
embedding is now hard as wax. Is there any way to deparaffinize processed
organs and may I embed them in OCT for proper cryosectioning? I imagine
that the liver would get dehydrated, I would get crappy sections, and Oil
Red O won't work.

Any suggestions are welcome.

Thank you so much,

Zoe W.


-- 
It costs nothing to say something kind. Even less to shut up altogether.

    --Nathan Fillion
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[Histonet] help with dead fungi

[Bartlett, Jeanine (CDC/OID/NCEZID)]

Good morning everyone,

Does anyone have  a protocol they use to demonstrate dead fungi in FFPPE tissue?

Thanks!

Jeanine H. Bartlett, BS HT(ASCP), QIHC
Centers for Disease Control and Prevention
Infectious Diseases Pathology Branch
1600 Clifton Road, NE
MS/G-32
Atlanta, Ga 30333
404-639-3590
jeanine.bartl...@cdc.hhs.gov

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Re: [Histonet] help with dead fungi

[Rene J Buesa]

Do you mean to try to demonstrate if the fungi you find in a sample WAS dead or 
alive before it was killed during the fixation and processing?
My take on this is the following: if a fungi dies naturally in a tissue 
(either lung or nail, or whatever) that dead fungi either is decayed and lost 
or its shape has to be modified in a way that it can be microscopically 
distinguished by the pathologist from other alive fungi.
On the other hand you could try to use DNA or RNA staining to localize the 
nuclei and decide if the structure seems to correspond to that of an alive 
fungi.
I think that if you find fungal structures in abundance in a FFPE tissue they 
should correspond to living fungi at the moment the FFPE process started.
René J.



From: Bartlett, Jeanine (CDC/OID/NCEZID) j...@cdc.gov
To: 'histonet@lists.utsouthwestern.edu' histonet@lists.utsouthwestern.edu 
Sent: Thursday, October 11, 2012 7:53 AM
Subject: [Histonet] help with dead fungi

Good morning everyone,

Does anyone have  a protocol they use to demonstrate dead fungi in FFPPE tissue?

Thanks!

Jeanine H. Bartlett, BS HT(ASCP), QIHC
Centers for Disease Control and Prevention
Infectious Diseases Pathology Branch
1600 Clifton Road, NE
MS/G-32
Atlanta, Ga 30333
404-639-3590
jeanine.bartl...@cdc.hhs.gov

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[Histonet] help ! Dako Seymour labeling software - autostainer plus

[richard wild]

Hello dear histoneters

First let me thank you all for your help for lost password (autostainer 
plus software) -  a - a tip is ok.


I am begining to test the autostainer and now eveything seems fine 
(there was a stuck tube but I could settle the problem)


_Corrupted files : Dako Seymour software (TLP 2742 printer)_

   Autostainer plus can print labels directly from main software.
   But printing labels can be done from another computer if you use a
   another provided software (Dako Seymour labeling software)

   My software dako Seymour 5.0  is on two floppies and two compressed
   files are corrupted */VB40016.DL_/* (on first floppy) and
   */ARW01DN0.IC_/* (on second floppy)
   _It would be great if someone could send me by mail the two missing
   working files_ (compressed format, exactly as I have written them
   here would be better, but may be uncompressed files that are in the
   computer could work - these files are not very heavy and can easily
   be sent)

   I asked Dako France but they dont have anymore the floppies of the
   Dako Seymour labeling software (5.0)

_Does someone have a list or a link of best protocols (procedures) for 
the machine ?_


have a nice day
Best regards

R Wild
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[Histonet] Help ! autostainer plus - lost name and password

[richard wild]

Hello

I just bought a second hand Dako autostainer from Germany (I live in 
France) from a broker.
The machine looks fine but I can't test it because_I don't have identity 
and password _to get inside the software.


Does anyone know how if it is possible to localise the password and 
identities in the hard disk (some ini file ? by the regedit ? ... but 
where ?) ?
Or is it possible to destroy a file, so that the software begins a new 
cycle asking password and all ?

Or is there any other procedure to get through ?

DAKO autostainer plus or LABVISION 480 could use similar software.

Best friendly regards.

Richard
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Re: [Histonet] help ! paraffin section

[Rene J Buesa]

What you describe is a typical example of poor paraffin infiltration = the 
paraffin has not infiltrated the tissue and when you prepare the final block it 
will consist of 2 different components; the tissue and the paraffin. That is 
why you end with a good paraffin section without the tissue.
Poor paraffin infiltration is always caused by an improper sequence while 
tissue processing. Either the fixation is incomplete 
OR the dehydration is incomplete and there is water in the tissue when you go 
to the clearing stage 
OR the clearing stage is incomplete and the tissue still has alcohol 
(immiscible with paraffin) when the tissue goes to the paraffin 
OR the paraffin infiltration is too short.
The problem resides in your processing protocol and there is nothing you can do 
about that at the end.
Try to check your processing protocol to eliminate the problem.
If this is happening all of the sudden while you used to have good results 
previously, then you either have changed reagents or the reagents are not in a 
good condition.
René J. 



From: Megha Kumar meg...@g.clemson.edu
To: histonet@lists.utsouthwestern.edu 
Sent: Tuesday, August 7, 2012 11:45 PM
Subject: [Histonet] help ! paraffin section

Hi All
I am trying to section adult mouse intestine and skin using paraffin
embedding. However, when i section, the tissue is torn although the rest of
the paraffin looks perfect. Please suggest why this is happening. Also,
sometimes the skin sections fall off the slides when I perform in situ
hybridization. Any ideas how to prevent this?
Please help! i am a beginner in histology and dont' know what to do!
regards
Megha


*
*
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Re: [Histonet] help ! paraffin section

[Mehmet Fatih BOZKURT]

In addition to Rene's comment,to cut coagulated tissue (skin that have
new wound crust) and calcified tissue is difficult.

On Wed, Aug 8, 2012 at 6:45 AM, Megha Kumar meg...@g.clemson.edu wrote:

 Hi All
 I am trying to section adult mouse intestine and skin using paraffin
 embedding. However, when i section, the tissue is torn although the rest of
 the paraffin looks perfect. Please suggest why this is happening. Also,
 sometimes the skin sections fall off the slides when I perform in situ
 hybridization. Any ideas how to prevent this?
 Please help! i am a beginner in histology and dont' know what to do!
 regards
 Megha


 *
 *
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-- 
Mehmet Fatih BOZKURT, DVM, PhD
Afyon Kocatepe University
Faculty of Veterinary Medicine
Department of Pathology
03030, ANS Campus
Afyonkarahisar-TURKEY
Tel: +902722281312-173/237
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[Histonet] help ! paraffin section

[Megha Kumar]

Hi All
I am trying to section adult mouse intestine and skin using paraffin
embedding. However, when i section, the tissue is torn although the rest of
the paraffin looks perfect. Please suggest why this is happening. Also,
sometimes the skin sections fall off the slides when I perform in situ
hybridization. Any ideas how to prevent this?
Please help! i am a beginner in histology and dont' know what to do!
regards
Megha


*
*
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Re: [Histonet] Help

[Brendal Finlay]

Nancy,


We've had similar issues with fatty tissue falling off of the slides
while performing IHC.  We use Superfrost + slides which we have found
to really hold the tissue well.  Also, I have learned through reading
round on the Histonet that air drying doesn't completely remove the
water from the middle area of a tissue section.  For this reason, we
no longer air dry at all unless it's a slide that was cut the day
before and just happened to be air dried.  


Our protocol changed to cutting the slides and draining them well,
then putting them in a 60 C oven for 15 minutes.  Then the slides
are run down to water on an automated stainer with another 15 minute
time in the oven on the stainer.  


A specific instance when the tissue falls off, was during antigen
retrieval in Trilogy in a pressure cooker.  If the pressure was
manually released, this would cause the Trilogy to boil and it would
separate the tissue from the slide.  Ourprotocol changed to 12
minutes in the pressure cooker with Trilogy, then around 8 minutes to
wait for the pressure to release on it's own.  We would then rinse
softly in distilled water to remove the Trilogy.  This also seemed
to help with the issue.

  

The combination of this has worked fairly well for us with some
exceptionally stubborn tissue still attempting to fall off of the
slides.  I would love to hear of other's experiences and how they
resolved this.  


I do wonder about the length of time in your oven.  I had spoken with
one of our Biocare reps about this when we encountered the problem and
he felt that longer than 30 minutes in the oven would damage the
specimen's IHC integrity.  


Brendal Finlay HT (ASCP)


Original message-
From: Cloughley-Gray, Nancy cloughl...@rvh.on.ca
Date: Fri, 18 May 2012 14:02:35 -0500
To:
'histonet@lists.utsouthwestern.edu'histonet@lists.utsouthwestern.edu
Subject: [Histonet] Help

 I'm a Histotechnologist working in the Regional Hospital in Barrie,
ON Canada. We are using the Ventana Ultra for our Immunohistochemistry
(IHC). Since the end of February, we have been having issues with some
tissues lifting off our positive (marked with +) charged slides. It
seems to be mostly with the fatty and/or larger sections. We now dry
our slides for one hour at room temperature (R.T.) and an additional
hour at 60 degrees C. We cut our IHC sections at 4 um. Since we have
tried 2 different types of + slides and will be trying another type of
charged slide (from Newcomer this time) I was wondering if anyone has
any other suggestions?
 I also have another question regarding a QC (quality control) issue.
We use a multi-tissue control that is applied to the top of all our
test slides for IHC. One of our paths commented that there is some
positive staining in the smooth muscle nuclei of thenormal bowel when
we are testing for Progesterone (PR). We are using a Heat Induce
Epitope Retrieval (HEIR) of 36 minutes with CC1 (Ventana's proprietary
buffer @ pH of 8.0-8.5) and a primary antibody incubation time of 16
minutes with PR clone 1E2. (Ventana instrumentation provides
pre-diluted antibodies and the user adjusts the concentration of the
antibody by adjusting the time the primary antibody is incubated with
the tissue).
 I am concerned about the implications of this staining and I have
not been able to find a reference to this kind of unusual staining
pattern. The bowel tissue that we are using as QC is from a 62 year
old female patient. I was wondering if anyone has had any experience
with this kind of staining and /or any references that I could use.
 
 Thanking you in advance,
 I look forward to your input,
 Nancy Cloughley-Gray MLT
 


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RE: [Histonet] Help

[Monfils, Paul]

I realize that such + slides come with the instruction to completely
dry slides at room temperature before placing in the drying oven.  I
have used these slides for many years, and have found this procedure to
be not only unnecessary, but sometimes problematic. I believe sections
are more likely to detach if dried at room temperature prior to oven
drying.  If the section is not lying perfectly flat against the glass -
and some types of tissue never do initially - room temperature drying
doesn't allow wrinkled areas or other problem areas to effectively
spread flat. Points that are in contact with the glass bond
electrostatically, but points that are separated from the glass, even by
a few microns, do not.  Then, when placed into the oven, such raised
areas cannot spread flat because closely adjacent areas are already
bonded to the slide, and cannot move.

After picking up sections from the waterbath, I allow them to stand
vertically and drain for no more than 5 minutes, then place them into
the drying oven at 70 degrees C. for an hour.  I very seldom have any
detachment problems with this protocol.

Paul M.


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[Histonet] Help

[Cloughley-Gray, Nancy]

I'm a Histotechnologist working in the Regional Hospital in Barrie, ON Canada. 
We are using the Ventana Ultra for our Immunohistochemistry (IHC). Since the 
end of February, we have been having issues with some tissues lifting off our 
positive (marked with +) charged slides. It seems to be mostly with the fatty 
and/or larger sections. We now dry our slides for one hour at room temperature 
(R.T.) and an additional hour at 60 degrees C. We cut our IHC sections at 4 um. 
Since we have tried 2 different types of + slides and will be trying another 
type of charged slide (from Newcomer this time) I was wondering if anyone has 
any other suggestions?
I also have another question regarding a QC (quality control) issue. We use a 
multi-tissue control that is applied to the top of all our test slides for IHC. 
One of our paths commented that there is some positive staining in the smooth 
muscle nuclei of the normal bowel when we are testing for Progesterone (PR). We 
are using a Heat Induce Epitope Retrieval (HEIR) of 36 minutes with CC1 
(Ventana's proprietary buffer @ pH of 8.0-8.5) and a primary antibody 
incubation time of 16 minutes with PR clone 1E2. (Ventana instrumentation 
provides pre-diluted antibodies and the user adjusts the concentration of the 
antibody by adjusting the time the primary antibody is incubated with the 
tissue).
I am concerned about the implications of this staining and I have not been able 
to find a reference to this kind of unusual staining pattern. The bowel tissue 
that we are using as QC is from a 62 year old female patient. I was wondering 
if anyone has had any experience with this kind of staining and /or any 
references that I could use.

Thanking you in advance,
I look forward to your input,
Nancy Cloughley-Gray MLT

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Re: [Histonet] Help

[Rene J Buesa]

As to your issue of tissue not adhering to the slides, you could try to check 
the expiration date of your (+) slides. Perhaps it is just an issue with the 
slide.
As to controlling the concentration of an antibody by changing the incubation 
time, that is somewhat unorthodox to say the least. You modify a concentration 
with dilution, not with time. Perhaps you could modify the HIER step, or try to 
dilute the antibody.
René J.

--- On Fri, 5/18/12, Cloughley-Gray, Nancy cloughl...@rvh.on.ca wrote:


From: Cloughley-Gray, Nancy cloughl...@rvh.on.ca
Subject: [Histonet] Help
To: 'histonet@lists.utsouthwestern.edu' histonet@lists.utsouthwestern.edu
Cc: Callan, Lisa call...@rvh.on.ca
Date: Friday, May 18, 2012, 4:02 PM


I'm a Histotechnologist working in the Regional Hospital in Barrie, ON Canada. 
We are using the Ventana Ultra for our Immunohistochemistry (IHC). Since the 
end of February, we have been having issues with some tissues lifting off our 
positive (marked with +) charged slides. It seems to be mostly with the fatty 
and/or larger sections. We now dry our slides for one hour at room temperature 
(R.T.) and an additional hour at 60 degrees C. We cut our IHC sections at 4 um. 
Since we have tried 2 different types of + slides and will be trying another 
type of charged slide (from Newcomer this time) I was wondering if anyone has 
any other suggestions?
I also have another question regarding a QC (quality control) issue. We use a 
multi-tissue control that is applied to the top of all our test slides for IHC. 
One of our paths commented that there is some positive staining in the smooth 
muscle nuclei of the normal bowel when we are testing for Progesterone (PR). We 
are using a Heat Induce Epitope Retrieval (HEIR) of 36 minutes with CC1 
(Ventana's proprietary buffer @ pH of 8.0-8.5) and a primary antibody 
incubation time of 16 minutes with PR clone 1E2. (Ventana instrumentation 
provides pre-diluted antibodies and the user adjusts the concentration of the 
antibody by adjusting the time the primary antibody is incubated with the 
tissue).
I am concerned about the implications of this staining and I have not been able 
to find a reference to this kind of unusual staining pattern. The bowel tissue 
that we are using as QC is from a 62 year old female patient. I was wondering 
if anyone has had any experience with this kind of staining and /or any 
references that I could use.

Thanking you in advance,
I look forward to your input,
Nancy Cloughley-Gray MLT

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[Histonet] Help with TRAP staining protocol

[Dave D Kojic]

Hello All,

I am about to start working on my MS thesis and working on ground section of 
the mandible with some implants and HA particulates , so I need some help to 
tap into some of your knowledge on TRAP staining for osteoclasts. I would 
appreciate if I can get any help with a TRAP staining protocol for the ground 
section of the bone. 

Thank you very much






Dave D Kojic DDS
Biomaterials Resident
Department of Prosthodontics
UAB School of Dentistry
614-1919 7th Ave S
Birmingham, AL 35294
Phone: 205.996.5746
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Re: [Histonet] help

[koellingr]

Hi Lydia, 

I think Tony Henwood has it exactly right in talking of DAB intensification.  
The article he sites and several others show how much more sensitive DAB 
intensification can make an ordinary iron reaction.  And you are not looking in 
bone marrow or spleen but somewhere where there is so little iron. 

  

More than once I've been burned by research colleagues who gave me tissue 
saying x should be upregulated and spend weeks looking for it until they say 
oop's, sorry-my bad. Intra-dermal injections can become sub-dermal. IP 
injections can be sub-optimal with rough handling of mice. IV tail vein 
injections can be missed. And MPTP is toxic and dangerous enough to work with 
that I'd be fumbling around and nervous myself.  If you are confident the model 
is working by some secondary marker such as tyrosine hydroxylase 
immunoreactivity then you are still are looking for minute quantities of iron. 

  

Several easily available papers on that very mouse model tell you the limited 
cell population to look for (one says-NOT in the big cells), and in a very 
specific region using coronal sections (I always used a mouse brain mold to 
section to be sure of the anatomical location) and several of the papers use 
classical iron histochemistry followed by DAB intensification and their 
procedures are in material and methods. 

  

Reaction might be working after all-just have to focus in on very limited 
reaction product.  Good luck hunting. 

  

Ray 

  

Ray Koelling 

PhenoPath Labs 

Seattle, WA 



- Original Message -




From: Lydia Gunawan lydia.guna...@unimelb.edu.au 
To: Histonet@lists.utsouthwestern.edu 
Sent: Tuesday, November 15, 2011 2:00:32 PM 
Subject: [Histonet] help 

Hi there, I am having trouble with Turnbull staining. Anybody can help me? 
As I want to detect and quantify iron in the brain for Parkinson experiment, I 
am using mice(C57Bl6)  brain that were treated with MPTP injection.  So, I have 
been trying to stain those brain using paraffin section with Turnbull blue but 
I have no luck. 
FYI, I have been using 7% of Potassium ferricyanide in 3% HCL and I incubated 
for 2 hours, 37'. I also did incubation in triton-x and H2O2 too but still not 
getting any iron on my sections. Could anyone help me to solve my problem? 
Thanks 



Lydia Gunawan 
Oxidation Biology Laboratory 
Mental Health Research Institute 
Melbourne Brain Centre 
Corner Royal Pde and Genetics Lane 
University of Melbourne, Level 4 
Parkville, Vic 3010 
email: lydia.guna...@unimelb.edu.au 


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Re: [Histonet] help

[John Kiernan]

 
 
 
 
 
 
 

The Turnbull's blue method as you describe it will not detect iron in tissues. 
 
Potassium ferricyanide will give a blue precipitate only with iron(II) 
(ferrous). In tissues, the iron is present as iron(III) (ferric) in such 
proteins as ferritin and haemosiderin. The iron of haemoglobin, though 
abundant, cannot be released by acid to react as Fe(II) or Fe(III) ions. The 
easiest way to detect Fe(III) is with potassium ferrocyanide - 0.05M in 0.2M 
HCl, for 30m - which gives a Prussian blue deposit (Perls' reaction).  If you 
want to do a Turnbull's blue method, which some say is a bit more sensitive, 
you must first reduce all the Fe(III) in the tissue to Fe(II) with dilute 
ammonium sulphide. All the iron then ends up as precipitated FeS, which will 
react with an acidified potassium ferricyanide solution to produce Turnbull's 
blue.
 
In fact, Prussian and Turnbull's blues are the same compound (see inorganic 
chemistry textbooks). A faint or invisible reaction product can be amplified 
because this pigment behaves like peroxidase, catalyzing the oxidation of DAB 
by H2O2 (see e.g. Connor et al. 1995). The sensitivity can be further increased 
with chemical tricks to change the brown oxidation product of DAB into larger 
quantities of black stuff (Moos  Mollgard 1993).
 
References. 
Connor JR et 4 al (1995) A histochemical study of iron-positive cells in the 
developing rat brain. J. Comp. Neurol. 355:111-123. 
Moos T  Mollgard K (1993) A sensitive post-DAB enhancement technique for 
demonstration of iron in the central nervous system. Histochemistry 99:471-475. 
 
John Kiernan 
Anatomy, UWO 
London, Canada 
= = =

- Original Message -
From: Lydia Gunawan lydia.guna...@unimelb.edu.au 
To: Histonet@lists.utsouthwestern.edu 
Sent: Tuesday, November 15, 2011 2:00:32 PM 
Subject: [Histonet] help 

Hi there, I am having trouble with Turnbull staining. Anybody can help me? 
As I want to detect and quantify iron in the brain for Parkinson experiment, I 
am using mice(C57Bl6)  brain that were treated with MPTP injection.  So, I have 
been trying to stain those brain using paraffin section with Turnbull blue but 
I have no luck. 
FYI, I have been using 7% of Potassium ferricyanide in 3% HCL and I incubated 
for 2 hours, 37'. I also did incubation in triton-x and H2O2 too but still not 
getting any iron on my sections. Could anyone help me to solve my problem? 
Thanks 



Lydia Gunawan 
Oxidation Biology Laboratory 
Mental Health Research Institute 
Melbourne Brain Centre 
Corner Royal Pde and Genetics Lane 
University of Melbourne, Level 4 
Parkville, Vic 3010 
email: lydia.guna...@unimelb.edu.au 


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[Histonet] help

[Lydia Gunawan]

Lydia Gunawan
Oxidation Biology Laboratory
Mental Health Research Institute
Melbourne Brain Centre
Corner Royal Pde and Genetics Lane
University of Melbourne, Level 4
Parkville, Vic 3010
email: lydia.guna...@unimelb.edu.au


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[Histonet] help

[Lydia Gunawan]

Hi there, I am having trouble with Turnbull staining. Anybody can help me?
As I want to detect and quantify iron in the brain for Parkinson experiment, I 
am using mice(C57Bl6)  brain that were treated with MPTP injection.  So, I have 
been trying to stain those brain using paraffin section with Turnbull blue but 
I have no luck.
FYI, I have been using 7% of Potassium ferricyanide in 3% HCL and I incubated 
for 2 hours, 37'. I also did incubation in triton-x and H2O2 too but still not 
getting any iron on my sections. Could anyone help me to solve my problem? 
Thanks



Lydia Gunawan
Oxidation Biology Laboratory
Mental Health Research Institute
Melbourne Brain Centre
Corner Royal Pde and Genetics Lane
University of Melbourne, Level 4
Parkville, Vic 3010
email: lydia.guna...@unimelb.edu.au


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[Histonet] Help

[Corrie Vernick]

I am currently a histology student at Keiser University. I am doing a project 
for my routine staining class about Celestine Blue. I've been able to find 
information on why it was created, the chemical make up, and some of it's uses 
including the trichrome stain. I am having trouble finding images of slides 
stained with Celestine Blue. Any additional information about the uses would be 
helpful as well! Thank you, Corrinne VernickKeiser UniversityFL U.S.A   
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[Histonet] Help finding metal embedding molds

[McLaughlin, Terry]

Hello All,
This is my first time posting a request. We use metal embedding molds,
and would like to order one particular size. The outer measurements are
2.5mm x 2.5mm x 
0.6 mm. We are wondering if anyone has approximately 40-50 of them we
could purchase-new or used. The reason we need this size is because most
of our research tissue fits perfectly into this size and depth. We have
18 of them and quite often need 30-60 for a particular project. We found
them from one of the vendors, but the price is quite expensive. We are
hoping someone out there can help us.
 
Much Thanks,
Terry McLaughlin 
Histology Specialist 
AI DuPont Hospital for Children 
1600 Rockland Rd. 
Wilmington, DE. 19803 
phone 302-651-6771 
fax-302-651-5010 

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[Histonet] Help with Masson's Trichrome - not working on cryo sections

[Jennifer Fricton]

Hello,

I am completely stumped by a problem we¹ve been having with our Masson¹s
Trichrome.  It isn¹t working properly on cryo sections.  We developed our
protocol several years ago and it has been a standard in our lab without any
trouble, until about four months ago when it just stopped working on frozen
sections.  It still works fine on paraffin sections.

You can see images at this link:
http://www.med.umn.edu/lhi/prod/groups/med/@pub/@med/@dom/@lhi/documents/con
tent/med_content_340494.pdf

What seems to be happening is that the beibrich scarlet + acid fuchsin
reagent is either not staining the muscle tissue, or is washing out of the
tissue.  The aniline blue is still staining the connective tissue very well
(it stopped working, too, but we solved by reducing time in water rinse
following stain).  My Masson¹s protocol is very standard ­ essentially
straight out of the Armed Forces Institute of Pathology Manual (p. 94).  I
fix my cryosections in 10% NBF x 30 mins. before starting the protocol.
I¹ve made sure my formalin is fresh and properly stored, with no white
precipitate.  I¹ve tried the Bouin¹s mordant step at both 56C and room temp
overnight.  I¹ve reduced my distilled water rinses following the biebrich
scarlet and aniline blue steps to just a quick dip, thinking maybe I was
de-staining too much before setting the stains with acetic acid.  I¹ve
re-made all solutions, checked all components and pH levels.  I haven¹t done
anything different with my frozen tissues, and I¹ve tried several different
freshly cut tissues.

Has anyone seen this before and have any tips on what is going wrong?  Your
help is greatly appreciated!

-- 
Jennifer L. Fricton, B.S.
Scientist, Lab Manager
Lillehei Heart Institute Histology  Microscopy Core Facility
University of Minnesota School of Medicine, Division of Cardiology
312 Church Street SE
4-290 Nils Hasselmo Hall
Minneapolis, MN  55455
Lab: 612-626-3090

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