[Histonet] Freezing methode

Tue Dec 11 13:02:21 CST 2007

Hello M. Reinders,
One method to snapfreeze brains without much damages is to freeze into an
isopentane bath cooled at -40°C. You may want to have a look at the
Snapfrost machine (www.alphelys.com, section Specimen freezing) that
controls precisely temperature and ensures a good and reproducible quality. 
Kind regards
Pierre

You wrote : 
Message: 7
Date: Mon, 10 Dec 2007 17:19:39 +0100
From: "Reinders, N.R. (Niels)" <N.R.Reinders <@t> uu.nl>
Subject: [Histonet] Freezing methode
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID:

<95D29B889F8EA44498550ECAA1C2763D0139E5FA <@t> uu01msg-exb02.soliscom.uu.nl>

Content-Type: text/plain;	charset="us-ascii"

 Hello histosearch,

I was wondering if anyone ever heard about a freezingmethode where a whole
mouse-brain is placed is a cooled (nitrogen, dry-ice or
methylbutane) brain matrix? Will it freeze to it not to be removed in one
piece anymore? Or will it still change its shape in a matrix? I need an
alternative because I want less-experienced people to be able to freeze the
brain without much damage (I lost some brain lately). All idea's are
welcome!

Kind regards, N.Reinders

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Today's Topics:

   1. Processing schedule for mouse tissue. (Colin Nixon)
   2. Re: Processing schedule for mouse tissue. (Kim Merriam)
   3. Image analysis sample size (McGinley,John)
   4. tissue burnt/dried on processor (curt tague)
   5. Cleaning Paraffin From Floors (Schaundra Walton)
   6. IHC anti-vimentin on mouse tissue (Perry, Samuel)
   7. Freezing methode (Reinders, N.R. (Niels))
   8. RE: tissue burnt/dried on processor (Kemlo Rogerson)
   9. Job Opportunity in Silicon Valley - Histo Tech (Majid ghoddusi)
  10. CD11c on paraffin (Andrew Wang)
  11. Re: Image analysis sample size (Bob Nienhuis)

----------------------------------------------------------------------

Message: 1
Date: Mon, 10 Dec 2007 08:30:56 -0000
From: "Colin Nixon" <c.nixon <@t> beatson.gla.ac.uk>
Subject: [Histonet] Processing schedule for mouse tissue.
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID:

<0E5DFBD0E5E27443A0987F7664787163BAC890 <@t> exchange-be4.centre.ad.gla.ac.uk>

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Hi,

I was wondering if anyone could share there processing schedules for mouse
tissue. My current schedule is below but I feel I may be over processing the
tissue to some degree. The tissue is processed overnight on a ThermoFisher
Excelsior processor.

70% Alcohol 35 mins
90% Alcohol 40 mins
95% Alcohol 45 mins
100% Alcohol 60 mins
100% Alcohol 60 mins
100% Alcohol 60 mins
Xylene 45 mins
Xylene 60 mins
Xylene 60 mins
Wax 90 mins
Wax 90 mins
Wax 90 mins

many thanks,

Colin 

------------------------------

Message: 2
Date: Mon, 10 Dec 2007 05:10:21 -0800 (PST)
From: Kim Merriam <kmerriam2003 <@t> yahoo.com>
Subject: Re: [Histonet] Processing schedule for mouse tissue.
To: Colin Nixon <c.nixon <@t> beatson.gla.ac.uk>,
	histonet <@t> lists.utsouthwestern.edu
Message-ID: <114727.62674.qm <@t> web50304.mail.re2.yahoo.com>
Content-Type: text/plain; charset=us-ascii

I have a VIP, so I don't know about your specific processor; but I would
decrease your times to about 30 minutes per station for the first 3
alchohols and to a total of about 90 minutes for the 100% alchohol, xylene
and paraffin (for example, 30-30-30, or something like that).  I am sure
someone with your processor can chime in with a more specific mouse
schedule.

Good luck!
Kim

Kim Merriam, MA, HT(ASCP)
Cambridge, MA

----- Original Message ----
From: Colin Nixon <c.nixon <@t> beatson.gla.ac.uk>
To: histonet <@t> lists.utsouthwestern.edu
Sent: Monday, December 10, 2007 3:30:56 AM
Subject: [Histonet] Processing schedule for mouse tissue.

Hi,

I was wondering if anyone could share there processing schedules for mouse
tissue. My current schedule is below but I feel I may be over processing the
tissue to some degree. The tissue is processed overnight on a ThermoFisher
Excelsior processor.

70% Alcohol 35 mins
90% Alcohol 40 mins
95% Alcohol 45 mins
100% Alcohol 60 mins
100% Alcohol 60 mins
100% Alcohol 60 mins
Xylene 45 mins
Xylene 60 mins
Xylene 60 mins
Wax 90 mins
Wax 90 mins
Wax 90 mins

many thanks,

Colin 

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Message: 3
Date: Mon, 10 Dec 2007 08:13:04 -0700
From: "McGinley,John" <John.McGinley <@t> ColoState.EDU>
Subject: [Histonet] Image analysis sample size
To: "histonet <@t> lists.utsouthwestern.edu"
	<histonet <@t> lists.utsouthwestern.edu>,	"ihcrg <@t> googlegroups.com"
	<ihcrg <@t> googlegroups.com>
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Hi,

I have a question regarding image analysis and sample size (microscopic
fields). Our lab has been estimating proliferation indices in IHC stained
(BrdU and Ki-67) rat mammary gland and mammary tumors by evaluating ten 400x
microscopic fields chosen at random, e.g. green dots placed on the tissue
section prior to review under the scope in order to prevent selection bias
toward heavily stained "hot spot" areas. We're using a CAS-200 imaging
system that measures total nuclear area and DAB percent positive area above
a specified threshold. The problem is that specimens can have a large
range/variance depending upon the areas chosen for analysis and sampling 10
fields may not be enough. I'm thinking the best way to compensate for this
problem would be to increase the number of fields until the variance
stabilizes. Does anyone have an algorithm like an Excel macro or something
similar that addresses this problem, i.e. will estimate how many more fields
to count beyond 10 based on current variance? Thanks in advance.

Regards,

John N. McGinley
Cancer Prevention Laboratory
Colorado State University
john.mcginley <@t> colostate.edu

------------------------------

Message: 4
Date: Mon, 10 Dec 2007 07:59:16 -0800 (PST)
From: curt tague <opiecurt <@t> yahoo.com>
Subject: [Histonet] tissue burnt/dried on processor
To: histonet <@t> lists.utsouthwestern.edu
Message-ID: <746070.67171.qm <@t> web81602.mail.mud.yahoo.com>
Content-Type: text/plain; charset=iso-8859-1

to summarize, we had some of the reagents errantly changed on the tissue
processor. some of the 100% alc. seem to have been changed with 95% which we
thing resulted in water in the tissue through the xylene and wax. however,
the tissue was really dry and brittle.... i would really love to resolve
this problem rather than tell the clients that their tissue is destroyed
beyond repair. i have a picture i can forward to you if anyone would like to
take a look. basically, the epi, on skins specifically, is staining really
light. almost like we didn't leave it in there long enough? pathologists
were not happy to say teh least.

  please help, let me know if i can forward the pic.

  curt

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Message: 5
Date: Mon, 10 Dec 2007 08:14:29 -0800 (PST)
From: Schaundra Walton <schaundrawalton <@t> yahoo.com>
Subject: [Histonet] Cleaning Paraffin From Floors
To: Histonet <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <751959.56571.qm <@t> web58909.mail.re1.yahoo.com>
Content-Type: text/plain; charset=iso-8859-1

Hey everybody!

  We are moving into a brand new lab in January 08.  I got my first look at
our new space last week and I'm concerned about the flooring they've
selected for the new histo lab.  It is vinyl. Which means we won't be able
to scrape the paraffin that inevitably collects on the floors with our razor
blade scraper.  Does anyone have any suggestions?  What are other people out
there doing to prevent paraffin build-up on the floors?

  Thanks in advance!

Schaundra Walton BS HTL(ASCP)
Swedish American Hospital
1401 E. State St. 
Rockford, IL 61104

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Message: 6
Date: Mon, 10 Dec 2007 11:16:37 -0500
From: "Perry, Samuel" <Samuel_Perry <@t> DFCI.HARVARD.EDU>
Subject: [Histonet] IHC anti-vimentin on mouse tissue
To: <histonet <@t> lists.utsouthwestern.edu>
Cc: "Chu, Gerald Chen,M.D.,Ph.D." <GCCHU <@t> PARTNERS.ORG>
Message-ID:
	<D3C984272CCAA741879C0B9339D1A7670B8FEA <@t> PHSXMB24.partners.org>
Content-Type: text/plain; charset="us-ascii"

Hi All,

I am having trouble finding a vimentin antibody that works well for IHC in
mouse tissue.  I have tried a goat anti-vimentin from Chemicon (AB1620) and
a rabbit monoclonal anti-vimentin from Epitomics (cat. # 4211-1).  The
Chemicon had a lot of non-specific staining and the Epitomics showed no
staining in mouse tissue.
If anyone knows of a good IHC staining anti-vimentin antibody (preferably of
rabbit specie) please let me know.  Thanks -Sam 

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Message: 7
Date: Mon, 10 Dec 2007 17:19:39 +0100
From: "Reinders, N.R. (Niels)" <N.R.Reinders <@t> uu.nl>
Subject: [Histonet] Freezing methode
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID:

<95D29B889F8EA44498550ECAA1C2763D0139E5FA <@t> uu01msg-exb02.soliscom.uu.nl>

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 Hello histosearch,

I was wondering if anyone ever heard about a freezingmethode where a whole
mouse-brain is placed is a cooled (nitrogen, dry-ice or
methylbutane) brain matrix? Will it freeze to it not to be removed in one
piece anymore? Or will it still change its shape in a matrix? I need an
alternative because I want less-experienced people to be able to freeze the
brain without much damage (I lost some brain lately). All idea's are
welcome!

Kind regards, N.Reinders

N.Reinders, molecular analist, 

University of Utrecht, NL

Yalelaan 2 3584 CM (363), Utrecht

(0031) (0)30 2533818

N.R.Reinders <@t> uu.nl <mailto:N.R.Reinders <@t> uu.nl> 

------------------------------

Message: 8
Date: Mon, 10 Dec 2007 16:21:45 -0000
From: "Kemlo Rogerson" <Kemlo.Rogerson <@t> waht.swest.nhs.uk>
Subject: RE: [Histonet] tissue burnt/dried on processor
To: "curt tague" <opiecurt <@t> yahoo.com>,
	<histonet <@t> lists.utsouthwestern.edu>
Message-ID:

<86ADE4EB583CE64799A9924684A0FBBF0222F321 <@t> wahtntex2.waht.swest.nhs.uk>
Content-Type: text/plain;	charset="us-ascii"

Odd that, if the tissue was not fully dehydrated then the xylene wouldn't
have fully penetrated nor would the paraffin penetrated; it ought to be
soggy not 'dry and burnt'.

You could try revitalising by soaking in oil of cedar wood then
reprocessesing but that's usually for 'overprocessed' rather than
'underprocessed' material. Interesting xylene, if that's what you use, can
usually accommodate some water especially if fresh and clean (the xylene
that is)and I have known tissue be successfully processed despite not using
100% and I wonder if the brittleness has been caused by something else; Too
much heat? 

Kemlo Rogerson
Pathology Manager
DD   01934 647057 or extension 3311
Mob 07749 754194; Pager 07659 597107;
The road to success is dotted with many tempting parking places.
--Author Unknown 

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------------------------------

Message: 9
Date: Mon, 10 Dec 2007 08:49:48 -0800
From: "Majid ghoddusi" <majid.ghoddusi <@t> gmail.com>
Subject: [Histonet] Job Opportunity in Silicon Valley - Histo Tech
To: histonet <@t> lists.utsouthwestern.edu
Message-ID:
	<f7650feb0712100849h7fb92fb1mc719ad4621ac976a <@t> mail.gmail.com>
Content-Type: text/plain; charset=ISO-8859-1

Comparative Biosciences Inc. has a full time position open for an
experienced Research Associate or Scientist with proficiency in *
Immunohistochemistry*.  This researcher in this position will also perform
in situ hybridization, cell culture and molecular techniques in of state of
the art GLP histology laboratory.  BS with demonstrable experience and
capability will be considered.  Flexible schedules are possible and our
benefits are outstanding. Salary: $40,000 to $60,000 per year depending on
qualifications and experience.

Please visit our website: http://www.compbio.com/job-openings.html

*Majid Ghoddusi, DVM, PhD*
*Veterinary Pathologist*
*Comparative Biosciences, Inc.*
*786 Lucerne Drive, *
*Sunnyvale, CA 94085*
*http://www.compbio.com/* <http://www.compbio.com/>
*Ph: 408-738-9265*
*Fax: 408-738-9278*

------------------------------

Message: 10
Date: Mon, 10 Dec 2007 12:22:23 -0500
From: "Andrew Wang" <andrew.wang <@t> tufts.edu>
Subject: [Histonet] CD11c on paraffin
To: histonet <@t> lists.utsouthwestern.edu
Message-ID:
	<6c0279d90712100922w4b6fe8ffnbdca8cafe9ae058 <@t> mail.gmail.com>
Content-Type: text/plain; charset=ISO-8859-1

Hi,

I am hoping to stain human tissue embedded in paraffin for CD11c.
Does anyone have any success with this protocol?  Any help would be great
for this newbie.

Andrew Wang MSIV
Department of Pathology
Tufts University Sackler School of Graduate Biomedical Sciences University
of Medicine and Dentistry of New Jersey Robert Wood Johnson Medical School

------------------------------

Message: 11
Date: Mon, 10 Dec 2007 09:36:50 -0800
From: "Bob Nienhuis" <bob.nienhuis <@t> gmail.com>
Subject: Re: [Histonet] Image analysis sample size
To: "McGinley,John" <John.McGinley <@t> colostate.edu>
Cc: "histonet <@t> lists.utsouthwestern.edu"
	<histonet <@t> lists.utsouthwestern.edu>,	"ihcrg <@t> googlegroups.com"
	<ihcrg <@t> googlegroups.com>
Message-ID:
	<45109da50712100936y1783be3p478a05a8a1084228 <@t> mail.gmail.com>
Content-Type: text/plain; charset=ISO-8859-1

You may want to do a sample power calculation.
See: <http://stat.ubc.ca/~rollin/stats/ssize/index.html>
for an online version.

Bob Nienhuis
UCLA / VA Medical Center
Los Angeles

On Dec 10, 2007 7:13 AM, McGinley,John <John.McGinley <@t> colostate.edu> wrote:

> Hi,
>
> I have a question regarding image analysis and sample size 
> (microscopic fields). Our lab has been estimating proliferation 
> indices in IHC stained (BrdU and Ki-67) rat mammary gland and mammary 
> tumors by evaluating ten 400x microscopic fields chosen at random, 
> e.g. green dots placed on the tissue section prior to review under the 
> scope in order to prevent selection bias toward heavily stained "hot 
> spot" areas. We're using a CAS-200 imaging system that measures total 
> nuclear area and DAB percent positive area above a specified 
> threshold. The problem is that specimens can have a large 
> range/variance depending upon the areas chosen for analysis and 
> sampling 10 fields may not be enough. I'm thinking the best way to 
> compensate for this problem would be to increase the number of fields 
> until the variance stabilizes. Does anyone have an algorithm like an 
> Excel macro or something similar that addresses this problem, i.e. will
estimate how many more fields to count beyond 10 based on current variance?
Thanks in advance.
>
> Regards,
>
> John N. McGinley
> Cancer Prevention Laboratory
> Colorado State University
> john.mcginley <@t> colostate.edu
>
>
>
>
>
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>

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