[Histonet] RE:Job Opportunity (Catherine Scott)

From:"Catherine Scott"


We (MPI Research) currently have an opening for Associate Scientist in
our plastics Lab. This person would be responsible for the day-to-day
operations in our newly built Plastics Lab.  Candidate must have 5+
years of histology experience along with (ASCP) HT/ HTL certification.
Candidate must have experience working with GMA, MMA, cardiac stents,
undecalcified bone, histomorphometry and special stains. The successful
candidate must be a self-starter, have leadership qualities and
troubleshooting abilities.
If interested please contact me a Catherine.scott@mpiresearch.com,
Also, for more information please check out our web site at
mpiresearch.com

Thank you,
Cathy

Catherine L Scott, B.A., HT (ASCP), ALAT
Associate Director, Pathology Services
54943 North Main Street
Mattawan, MI 49071-9399 USA
Telephone: 269.668-3336 ext. 1218
Email:Catherine.scott@mpiresearch.com






-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu
[mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of
histonet-request@lists.utsouthwestern.edu
Sent: Wednesday, January 26, 2005 1:15 PM
To: histonet@lists.utsouthwestern.edu
Subject: Histonet Digest, Vol 14, Issue 33

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Today's Topics:

   1. cd15 and morphology on bone marrows and lymph nodes
      (Van Eyck, Deb)
   2. reagent pricing (Jeff Birkner)
   3. RE: reagent pricing (Jeff Birkner)
   4. RE: reagent pricing (GUTIERREZ, JUAN)
   5. Re: Ache staining querry (John Kiernan)
   6. Rank-L (Patty Lott)
   7. Job opportunity central PA (Linda Hartman)
   8. Freezing Biopsies (Amos Brooks)
   9. Re: cd15 and morphology on bone marrows and lymph nodes
      (Andi Kappeler)
  10. Eluting Solution - Glycin-HCl (Melanie Black)
  11. RE: Fluorescence staining (C.M. van der Loos)
  12. testing connection (Carl Hobbs)
  13. subscribe to the histonet please, thanks (Wayne Holland)
  14. Hairy Cell Leukemia Spleen Tissue (Barnhart, Tammy)
  15. Attn: Roxanne Soto & Emily Smith (Greg Dobbin)
  16. Manual fixation/processing of reconstructed skin	cultures
      grown on polycarbonate filters (Colin Smith)
  17. Re: Freezing Biopsies (Kathleen Spencer)
  18. Re: cd15 and morphology on bone marrows and lymph nodes
      (Andi Kappeler)
  19. RMSF  (KarBieber@aol.com)
  20. Re: Freezing Biopsies (Gayle Callis)
  21. Re: RE: Fluorescence staining (Gayle Callis)
  22. Re: Freezing Biopsies (ajennings@unmc.edu)
  23. RE: Manual fixation/processing of reconstructed	skincultures
      grown on polycarbonate filters (Elizabeth Chlipala)
  24. mouse eye lens (Jarrin Miguel)


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

Message: 1
Date: Tue, 25 Jan 2005 12:59:04 -0600
From: "Van Eyck, Deb" 
Subject: [Histonet] cd15 and morphology on bone marrows and lymph
	nodes
To: "'histonet@lists.utsouthwestern.edu'"
	
Message-ID:
	
Content-Type: text/plain

	
     Hi all,  
1.I would really appreciate some replies to a couple of questions.  One
is
CD15; I thought  I had a pretty good antibody going, but it is still
problematic in some cases.  I would like to know what others are using
consistently with heme path approval.  The same goes for kappa and
lambda
and clonality. 
 

2.  The other issue is handling bone marrows and lymph nodes---we have
recently gotten rid of B-5 and we use NBF as our main fixative.  I would
like some replies in regards to what is the best fixation/decal
situation
for morphology purposes (does anyone feel that they have reached the b-5
nirvana state for good morphology) and still maintain immuno
staining????
Times and protocols and products would be great.    


Thanks----Deb Van Eyck-Anatomic Path
Waukesha Memorial Hospital
725 American Ave.
Waukesha,WI 53188
262-928-2112
fax:262-928-4849



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

Message: 2
Date: Tue, 25 Jan 2005 13:17:01 -0600
From: Jeff Birkner 
Subject: [Histonet] reagent pricing
To: "'histonet@lists.utsouthwestern.edu'"
	
Message-ID: 
Content-Type: text/plain

What is everyone paying for reagent alcohol, xylene and formalin?  

 

We are paying the following per gallon, not including shipping:

 

Reagent alcohol             $15.00

Xylene                           $28.00

10% NB Formalin           $5.45

 

Jeff Birkner, CT(ASCP)

Pathology Section Manager

Collaborative Laboratory Services, LLC

1005 Pennsylvania Ave.

Ottumwa, IA  52501

 

 



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

Message: 3
Date: Tue, 25 Jan 2005 13:56:47 -0600
From: Jeff Birkner 
Subject: [Histonet] RE: reagent pricing
To: "'histonet@lists.utsouthwestern.edu'"
	
Message-ID: 
Content-Type: text/plain

I will put the responses in an excel spreadsheet and share them with
anyone
that wants them.

 

  _____  

From: Jeff Birkner 
Sent: Tuesday, January 25, 2005 1:17 PM
To: 'histonet@lists.utsouthwestern.edu'
Subject: reagent pricing

 

What is everyone paying for reagent alcohol, xylene and formalin?  

 

We are paying the following per gallon, not including shipping:

 

Reagent alcohol             $15.00

Xylene                           $28.00

10% NB Formalin           $5.45

 

Jeff Birkner, CT(ASCP)

Pathology Section Manager

Collaborative Laboratory Services, LLC

1005 Pennsylvania Ave.

Ottumwa, IA  52501

 

 



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

Message: 4
Date: Tue, 25 Jan 2005 14:14:17 -0600
From: "GUTIERREZ, JUAN" 
Subject: RE: [Histonet] reagent pricing
To: "Jeff Birkner" ,
	
Message-ID:
	

Content-Type: text/plain;	charset="iso-8859-1"

Reagent alcohol	8.73/gal
Xylene		7.85/gal
Formalin		2.92/gal

Say Hi to Radar for me.

Juan C. Gutierrez, HT(ASCP)
Histology Laboratory Supervisor
(210)704-2533

My opinions are my own and do not reflect those of my employer.  Long
live free speech!


-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu
[mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of Jeff
Birkner
Sent: Tuesday, January 25, 2005 1:17 PM
To: 'histonet@lists.utsouthwestern.edu'
Subject: [Histonet] reagent pricing

What is everyone paying for reagent alcohol, xylene and formalin?  

 

We are paying the following per gallon, not including shipping:

 

Reagent alcohol             $15.00

Xylene                           $28.00

10% NB Formalin           $5.45

 

Jeff Birkner, CT(ASCP)

Pathology Section Manager

Collaborative Laboratory Services, LLC

1005 Pennsylvania Ave.

Ottumwa, IA  52501

 

 

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



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

Message: 5
Date: Tue, 25 Jan 2005 15:49:42 -0500
From: John Kiernan 
Subject: Re: [Histonet] Ache staining querry
To: Arvind Pundir 
Cc: histonet@lists.utsouthwestern.edu
Message-ID: <41F6B0E6.5B99D9E2@uwo.ca>
Content-Type: text/plain; charset=us-ascii

Assuming "Ache staining" is acetylcholinesterase activity
histochemistry, not a painful bruise:

The surface of the specimen will have been fixed more than 
the inside. AChE activity resists short fixation
in formaldehyde (such as overnight). Perhaps the outside
of the block has had more than is good for the enzyme.

In small specimens of animal tissues, both the choline 
esterases (and other esterases) are well preserved by 
overnight fixation in formaldehyde.
Human brains need much longer because of the large size,
and this can be expected to wreck enzyme activities. You
might still be able to stain the AChE protein with an 
immunohistochemical method.
-- 
-------------------------------
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1
   kiernan[AT]uwo.ca
   http://publish.uwo.ca/~jkiernan/
   http://instruct.uwo.ca/anatomy/530/index.htm
_______________________________
Arvind Pundir wrote:
> 
> i do Ache  staining frequently on human brain sections, but  facing
problem that there is no staining at the edges of my section can anyone
sort what it may be due to
> 
> 
> 
> thanks in advance for any suggestion or advice
> 
> 
> 
> Arvind Singh Pundir
> 
> National Brain Research Centre
> 
> INDIA
> 
>   ------------------------------------------------------------
> _______________________________________________
> Histonet mailing list
> Histonet@lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet



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

Message: 6
Date: Tue, 25 Jan 2005 16:30:45 -0600
From: Patty Lott 
Subject: [Histonet] Rank-L
To: 'histonet' 
Message-ID:
	<85F6C7A1330E794DB8540AFD001CC77E05328DA6@rosco.ortho.uab.edu>
Content-Type: text/plain

Does anyone know of an antibody for RANK-L to be used on FFPE mouse
bones?

 

Patty Lott, Laboratory Supervisor

Orthopaedic Research Laboratory 

Center for Metabolic Bone Disease Laboratory

University of Alabama at Birmingham

LHRB B37 0007

1919 7th Ave. South

Birmingham, AL 35294

(205) 934-2007

 



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

Message: 7
Date: Tue, 25 Jan 2005 20:38:45 -0500
From: "Linda Hartman" 
Subject: [Histonet] Job opportunity central PA
To: 
Message-ID: <002501c50347$c7335a80$1d04a245@linda47p53fn9e>
Content-Type: text/plain;	charset="iso-8859-1"

Mount Nittany Medical Center in State College, PA has an opening for a
full time histology technician.  The applicant will be responsible for
routine histology, frozen sections, special staining and IHC. 
 Mount Nittany Medical Center is a growing regional medical center
located mid-way between Philadelphia and Pittsburgh.  State College is
the home of the main campus of Penn State University.
Please check out our website at www.mountnittany.org
Linda Hartman HT(ASCP)
Histology section head
Mount Nittany Medical Center
State College, PA 16803
Phone: 814-234-6117
email: lhartman@mountnittany.org

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

Message: 8
Date: Tue, 25 Jan 2005 21:07:30 -0500
From: Amos Brooks 
Subject: [Histonet] Freezing Biopsies
To: histonet@lists.utsouthwestern.edu
Message-ID: <6.2.0.14.0.20050125210242.01da7198@pop.earthlink.net>
Content-Type: text/plain; charset="us-ascii"; format=flowed

Hi,
	I'm snap freezing kidney biopsies in OCT using isopentane that
is chilled 
with liquid nitrogen. I'm having a problem where the area that the
biopsy 
is gets raised up above the surface of the block. I'm not sure what is 
going wrong and more importantly what to do about it. Has anyone had 
similar problems and how did you solve it?

Thanks,
Amos Brooks





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

Message: 9
Date: Wed, 26 Jan 2005 07:52:43 +0100
From: "Andi Kappeler" 
Subject: Re: [Histonet] cd15 and morphology on bone marrows and lymph
	nodes
To: "Histonet" ,	"Van Eyck, Deb"
	
Message-ID: <002401c50373$a3254be0$27955c82@patho.unibe.ch>
Content-Type: text/plain;	charset="Windows-1252"

Hi Deb

We use mo-a-CD15, clone MMA, Becton-Dickinson 347420. Working conc. is
2.5
ug Ig/ml, pretreatment is HIER (citrate) in pressure cooker. The
'secret'
with this clone (and most other commercially available CD15 mAbs, such
as
C3D-1, BY87,  80H5, and more) is, that it is of Ig class IgM (and not
IgG1,
IgG2a, as most monoclonals that you commonly use). While most secondary
antibodies show some cross-reactivity with mAbs of  Ig class IgM,  you
have
to use a mouse-IgM specific secondary antibody to get optimal results
(e.g.
DakoCytomation E 0465, rb-a-mo IgM/Biotin). This applies also to a
couple of
other mAbs: they will work with 'regular' secondary antibodies, however,
they will work better with an IgM-specific secondary antibody. Therefore
it
is always useful to check the SpecSheet for the Ig-class of a given mAb.
If you are using a visualization system that comes from the supplier of
your
staining machine, you may have a problem, depending on the stainer. Some
of
them are obviously very reluctant to let you use secondary reagents of
your
choice... Then it is good to know, that immunos can be done manually ...
Hope this helps. Good luck.

Andi Kappeler
Insitute of Pathology, University of Bern, Switzerland



----- Original Message ----- 
From: "Van Eyck, Deb" 
To: 
Sent: Tuesday, January 25, 2005 7:59 PM
Subject: [Histonet] cd15 and morphology on bone marrows and lymph nodes


>
>      Hi all,
> 1.I would really appreciate some replies to a couple of questions.
One is
> CD15; I thought  I had a pretty good antibody going, but it is still
> problematic in some cases.  I would like to know what others are using
> consistently with heme path approval.  The same goes for kappa and
lambda
> and clonality.
>
>
> 2.  The other issue is handling bone marrows and lymph nodes---we have
> recently gotten rid of B-5 and we use NBF as our main fixative.  I
would
> like some replies in regards to what is the best fixation/decal
situation
> for morphology purposes (does anyone feel that they have reached the
b-5
> nirvana state for good morphology) and still maintain immuno
staining????
> Times and protocols and products would be great.
>
>
> Thanks----Deb Van Eyck-Anatomic Path
> Waukesha Memorial Hospital
> 725 American Ave.
> Waukesha,WI 53188
> 262-928-2112
> fax:262-928-4849
>
>
>
> This information is confidential and intended solely for the use of
the
> individual or entity to whom it is addressed.
> If you have received this email in error please notify the sender or
our
> Customer Support Center at (262) 928-2777.
>
> We have scanned this email and its attachments for malicious content.
> However, the recipient should check this email and any attachments for
the
> presence of viruses.
> ProHealth Care accepts no liability for any damage caused by any virus
> transmitted by this email.
>
> _______________________________________________
> Histonet mailing list
> Histonet@lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>




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

Message: 10
Date: Wed, 26 Jan 2005 09:21:12 +0200
From: Melanie Black 
Subject: [Histonet] Eluting Solution - Glycin-HCl
To: histonet@pathology.swmed.edu
Message-ID: 
Content-Type: text/plain; charset="us-ascii" ; format="flowed"

Hi There

Can anybody give me a method for making up Glycin-HCl(0.1M, pH 2.2) 
to be used an a blocking buffer in double immuno staining???

Many Thanks
Melanie
-- 
Cardiovascular Research Unit
Div. of Cardiothoracic Surgery
Chris Barnard Building
University of Cape Town
Anzio Road
Observatory
7925
Republic of South Africa

Tel +27 21 406-6589
Cel +27 82 469-3352
Fax +27 21 448-5935



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

Message: 11
Date: Wed, 26 Jan 2005 08:52:21 +0100
From: "C.M. van der Loos" 
Subject: [Histonet] RE: Fluorescence staining
To: histonet@lists.utsouthwestern.edu
Message-ID: <12c276c12c2991.12c299112c276c@amc.uva.nl>
Content-Type: text/plain; charset="us-ascii"


   Dear Eva,

   To my knowledge most of the traditional fluorochromes like FITC,
   rhodamine, doesn't stand dehydration at all. So in your case it fully
   depends on what type of fluorochrome you end up with after the TSA
   procedure.

   If you end up with FITC, the use of VectaShield
   ([1]www.vectorlabs.com) for mounting is highly recommended for
   the prevention of photobleaching.

   Chris van der Loos, PhD
   Dept. of Pathology
   Academical Medical Center M2-230
   Meibergdreef 9
   NL-1105 AZ Amsterdam
   The Netherlands

   phone:  +31 20 5665631
   fax:    +31 20 6960389
   e-mail: [2]c.m.vanderloos@amc.uva.nl


   ----- Original Message -----
                                  From 
   Eva C Anderson 
                                  Date 
   Tue, 25 Jan 2005 11:10:41 -0500
                                    To 
   histonet@lists.utsouthwestern.edu
                               Subject 
   [Histonet] Fluorescence staining

   Hi,
   We  are looking into trying some Fluorescence staining. We want to
try
   a  TSA  Fluorescence  System  from  PerkinElmer. The IHC protocol
does
   however leave me with some questions.
   We  always  dehydrate our samples after staining. The protocol
however
   doesn't mention this. It only says that the samples can be
   counterstained and mounted after incubation with the Fluorophore
   Tyramide. Should we dehydrate and is there a particular mounting
media
   we  should  use to prolong the time that fluorescence can be
detected?
   Any help would be greatly appreciated.
   Thanks in advance for all your help,
   Eva
   Georgetown University
   Research Assistant

References

   1. http://www.vectorlabs.com/
   2. mailto:c.m.vanderloos@amc.uva.nl


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

Message: 12
Date: Wed, 26 Jan 2005 08:09:43 -0000
From: "Carl Hobbs" 
Subject: [Histonet] testing connection
To: 
Message-ID: <000301c5037e$646dfea0$e8345c9f@Carlos>
Content-Type: text/plain; format=flowed; charset="iso-8859-1";
	reply-type=original

My emails keep getting bounced



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

Message: 13
Date: Wed, 26 Jan 2005 07:10:50 -0500
From: "Wayne Holland" 
Subject: [Histonet] subscribe to the histonet please, thanks
To: histonet@lists.utsouthwestern.edu
Message-ID: 
Content-Type: text/plain; format=flowed



_________________________________________________________________
Don't just search. Find. Check out the new MSN Search! 
http://search.msn.click-url.com/go/onm00200636ave/direct/01/




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

Message: 14
Date: Wed, 26 Jan 2005 07:29:02 -0600
From: "Barnhart, Tammy" 
Subject: [Histonet] Hairy Cell Leukemia Spleen Tissue
To: "Histonet (E-mail)" 
Message-ID: <1779904B5E82D511914C00D0B793339205BFD8B1@exchangent>
Content-Type: text/plain;	charset="iso-8859-1"

I am in need of the above control tissue.  We have not had a case here
that
we can find and would like to use it as a control for our TRAcP
antibody.
Willing to pay, hopefully not too much. Or, trade for other tissue :)
Thanks....

Tammy Barnhart, BS, HTL(ASCP)
Anatomic Pathology Supervisor
St. Alexius Medical Center
Bismarck, ND
tbarnhart@primecare.org
701-530-6730



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

Message: 15
Date: Wed, 26 Jan 2005 09:48:59 ADT
From: "Greg Dobbin" 
Subject: [Histonet] Attn: Roxanne Soto & Emily Smith
To: histonet@lists.utsouthwestern.edu
Message-ID: <41F7678B.22027.1B921F@localhost>
Content-Type: text/plain; charset=US-ASCII

Roxanne and Emily,
I am getting planning underway again for the Histonet Outpost. I 
sent out an e-mail to our `ad hoc' committee members yesterday 
and your e-mails bounced. Can you confirm that your e-mail 
addresses (below) are correct and if not, provide the proper one so 
I can forward you the e-mail again as well as all the replies I 
received to it.
Thanks.
Greg

ESmith@CuraGen.com
GREYTRUNK@aol.com


~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Greg Dobbin 
Research Technologist,
Pathology Lab,
Atlantic Veterinary College, U.P.E.I.
550 University Ave.
Charlottetown, P.E.I.
Canada,  C1A 4P3
Phone: (902)566-0744
Fax: (902)566-0851
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Happiness is a journey, not a destination.



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

Message: 16
Date: Wed, 26 Jan 2005 15:07:05 -0000
From: "Colin Smith" 
Subject: [Histonet] Manual fixation/processing of reconstructed skin
	cultures grown on polycarbonate filters
To: 
Message-ID:
	
<3E985CD1D7CA89499D1902BC3202AF3CC1A75E@uk-res-srv-03.thorspecialities.c
om>
	
Content-Type: text/plain;	charset="us-ascii"

Can anyone help?
I am currently setting up a basic histology system for analysing
reconstructed tissues grown in culture on polycarbonate filters, similar
to
Mattek's Epiderm. We currently have begged borrowed or stolen,
equipment-wise:
two water baths (ambient - 100C), a circulating warm air oven, a rocking
microtome and cold blocks, along with glass staining jars/racks, forceps
etc.
At the moment, equipment such as a wax dispenser, purpose built cooling
plates etc are out of the question until I can show that we can show
that the
model we are growing is validated, which will include
immunohistochemistry of
cytokeratin and cell adhesion markers etc. Thus we are in the catch 22
situation of having to prove something without the equipment needed to
do the
job, in order to justify buying the equipment!! But thats accountants
for
you, go figure.
Anyway, can anyone give any useful pointers/protocols for
fixing/processing/embedding/sectioning such tissues using such primative
equipment
Any help, no matter how trivial will be greatfully received.
 
Many Thanks in Advance,
 
Colin Smith BSc CBiol MIBiol Eurotox Registered Toxicologist
In Vitro Toxicologist 
Thor Specialities UK Ltd
Wincham Avenue,
Northwich
CW9 6GB
 
Tel: +44 1606 818873
Fax: +44 1606 818801
colins@thor.uk.com
 
 

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

Message: 17
Date: Wed, 26 Jan 2005 09:39:59 -0600
From: Kathleen Spencer 
Subject: Re: [Histonet] Freezing Biopsies
To: Amos Brooks 
Cc: histonet@lists.utsouthwestern.edu
Message-ID: <89982A33-6FB0-11D9-8BD6-000393967904@utmem.edu>
Content-Type: text/plain; charset=US-ASCII; format=flowed

Amos,
The way we did this was to place the biopsy in a plastic mold, add OCT, 
grab the huge forceps and plunge it into liquid nitrogen and count to 5 
or 10, I can't remember, but it always worked fine. We did not use 
isopentane. The surface of the block was always nice and smooth. It had 
to be! The biopsies were so tiny. What you can do is get a fresh mold, 
put a little OCT in the bottom, put the frozen block in and spray it a 
little with cryo spray. You should now have a smooth surface for 
sectioning.

Good Luck!

Kathleen Spencer HT (ASCP)
Lab Manager/LCM Supervisor
UTHSC


On Jan 25, 2005, at 8:07 PM, Amos Brooks wrote:

> Hi,
> 	I'm snap freezing kidney biopsies in OCT using isopentane that
is 
> chilled with liquid nitrogen. I'm having a problem where the area that

> the biopsy is gets raised up above the surface of the block. I'm not 
> sure what is going wrong and more importantly what to do about it. Has

> anyone had similar problems and how did you solve it?
>
> Thanks,
> Amos Brooks
>
>
>
> _______________________________________________
> Histonet mailing list
> Histonet@lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet




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

Message: 18
Date: Wed, 26 Jan 2005 17:18:31 +0100
From: "Andi Kappeler" 
Subject: Re: [Histonet] cd15 and morphology on bone marrows and lymph
	nodes
To: "Histonet" 
Message-ID: <006301c503c2$ad285ed0$27955c82@patho.unibe.ch>
Content-Type: text/plain;	charset="iso-8859-1"

We use the rb-a-mo IgM/Biotin in stead of our usual anti-mouse Ig
secondary
antibody (which is gt-a-mo Ig/Biotin). As we still stain manually (yes
that
exists...!) this is no problem. Adding the rb-a-mo IgM to your usual
secondary is theoretically possible, however if your secondary is a
'multi
link' reagent (anti-mouse and anti-rabbit cocktail) then you should not
add
a rb-a-mo IgM/Biotin as it will be bound by the anti-rb component of
your
'multi link' reagent. The system may still work, but I would prefer to
know
what I'm doing rather than to 'let something happen' inside a tube where
I
don't have control. If you want to add an anti-mouse IgM component to
your
'multi link', make sure it does not intefere with any of the components
that
is already in there (e.g. use a gt-a-mo IgM/Biotin).

Our protocol for CD15 looks as follows (in brief):
1. Deparaffinization --> TBS
2. Pretreatment, citrate buffer, pressure cooker, 7 min at 121 C, 1 bar;
cool down, wash
3. Incubate with mo-a-CD15, clone MMA, 2.5 ug Ig/ml
4. Wash
5. Incubate with rb-a-mo IgM (DakoCytomation E 0465, 1:500)
6. Wash
7. Incubate with enzyme system (ABC/HRP, LSAB+/HRP, whatever...)
8. Wash
9. Develop with 3,3'-diaminobenzidin /H2O2
10. Rinse, counterstain, mount

Best regards,
Andi

----- Original Message ----- 
From: "Van Eyck, Deb" 
To: "'Andi Kappeler'" 
Sent: Wednesday, January 26, 2005 4:20 PM
Subject: RE: [Histonet] cd15 and morphology on bone marrows and lymph
nodes


> thanks very much-------for the secondary system do you just use this
with
> another Dako product for example---are you using lsab or lsab +.   I
know
> some people use this system and just add the IgM to the
secondary--what
> specifically do you do.  this has been a real help Andi!
>
> > -----Original Message-----
> > From: Andi Kappeler [SMTP:kappeler@patho.unibe.ch]
> > Sent: Wednesday, January 26, 2005 12:53 AM
> > To: Histonet; Van Eyck, Deb
> > Subject: Re: [Histonet] cd15 and morphology on bone marrows and
lymph
> > nodes
> >
> > Hi Deb
> >
> > We use mo-a-CD15, clone MMA, Becton-Dickinson 347420. Working conc.
is
2.5
> > ug Ig/ml, pretreatment is HIER (citrate) in pressure cooker. The
'secret'
> > with this clone (and most other commercially available CD15 mAbs,
such
as
> > C3D-1, BY87,  80H5, and more) is, that it is of Ig class IgM (and
not
> > IgG1,
> > IgG2a, as most monoclonals that you commonly use). While most
secondary
> > antibodies show some cross-reactivity with mAbs of  Ig class IgM,
you
> > have
> > to use a mouse-IgM specific secondary antibody to get optimal
results
> > (e.g.
> > DakoCytomation E 0465, rb-a-mo IgM/Biotin). This applies also to a
couple
> > of
> > other mAbs: they will work with 'regular' secondary antibodies,
however,
> > they will work better with an IgM-specific secondary antibody.
Therefore
> > it
> > is always useful to check the SpecSheet for the Ig-class of a given
mAb.
> > If you are using a visualization system that comes from the supplier
of
> > your
> > staining machine, you may have a problem, depending on the stainer.
Some
> > of
> > them are obviously very reluctant to let you use secondary reagents
of
> > your
> > choice... Then it is good to know, that immunos can be done manually
...
> > Hope this helps. Good luck.
> >
> > Andi Kappeler
> > Insitute of Pathology, University of Bern, Switzerland
> >
> >
> >
> > ----- Original Message ----- 
> > From: "Van Eyck, Deb" 
> > To: 
> > Sent: Tuesday, January 25, 2005 7:59 PM
> > Subject: [Histonet] cd15 and morphology on bone marrows and lymph
nodes
> >
> >
> > >
> > >      Hi all,
> > > 1.I would really appreciate some replies to a couple of questions.
One
> > is
> > > CD15; I thought  I had a pretty good antibody going, but it is
still
> > > problematic in some cases.  I would like to know what others are
using
> > > consistently with heme path approval.  The same goes for kappa and
> > lambda
> > > and clonality.
> > >
> > >
> > > 2.  The other issue is handling bone marrows and lymph nodes---we
have
> > > recently gotten rid of B-5 and we use NBF as our main fixative.  I
would
> > > like some replies in regards to what is the best fixation/decal
> > situation
> > > for morphology purposes (does anyone feel that they have reached
the
b-5
> > > nirvana state for good morphology) and still maintain immuno
> > staining????
> > > Times and protocols and products would be great.
> > >
> > >
> > > Thanks----Deb Van Eyck-Anatomic Path
> > > Waukesha Memorial Hospital
> > > 725 American Ave.
> > > Waukesha,WI 53188
> > > 262-928-2112
> > > fax:262-928-4849
> > >
> > >
> > >
> > > This information is confidential and intended solely for the use
of
the
> > > individual or entity to whom it is addressed.
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> > > Customer Support Center at (262) 928-2777.
> > >
> > > We have scanned this email and its attachments for malicious
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> > > However, the recipient should check this email and any attachments
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> > the
> > > presence of viruses.
> > > ProHealth Care accepts no liability for any damage caused by any
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> > > transmitted by this email.
> > >
> > > _______________________________________________
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> > > Histonet@lists.utsouthwestern.edu
> > > http://lists.utsouthwestern.edu/mailman/listinfo/histonet
> > >
> >
> >
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>




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

Message: 19
Date: Wed, 26 Jan 2005 11:23:56 EST
From: KarBieber@aol.com
Subject: [Histonet] RMSF 
To: histonet@pathology.swmed.edu
Message-ID: <145.3e0c9baa.2f291e1c@aol.com>
Content-Type: text/plain; charset="US-ASCII"

Does anyone have a good source for an antibody for RMSF?

Thanks,
Karen


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

Message: 20
Date: Wed, 26 Jan 2005 09:25:16 -0700
From: Gayle Callis 
Subject: Re: [Histonet] Freezing Biopsies
To: Amos Brooks ,
	Histonet@lists.utsouthwestern.edu
Message-ID:
	<6.0.0.22.1.20050126085604.01af5e68@gemini.msu.montana.edu>
Content-Type: text/plain; charset="us-ascii"; format=flowed

Amos,

We experienced this too.  We solved by using Tissue Tek plastic
embedding 
molds 10 x 10 mm size.  Cut excess edges for a better heat sink around 
embedding well except for a small tab to hold onto.   Carefully pick a
mold 
that is not deformed already - look for the flat ones, not rounded!
Tissue 
Tek has newer round molds that do not require trimming, very nice, tidy
and 
FLAT!
Unfortunately, other brands of plastic molds we tried had thicker
plastic 
compared to Tissue Tek molds and blocks twisted out at freezing.

Coat bottom of mold with a THIN film of OCT.  Biopsy is laid out so it
is 
as flat as possible in OCT film on bottom.  Carefully, slowly - add more

OCT down corner of mold or away from top of biopsy to keep it from
floating 
up.  Overfilling with OCT results in cracked blocks; fill to top of
well.

  We tilt the beaker with cold isopentane , and introduce mold so bottom

makes contact with isopentant first.  When OCT starts to turn white ON 
BOTTOM,  immerse fully -about 10 sec or so.  Bottom freezing first
anchors 
biopsy and keeps it flat, no lift up syndrome.  If frozen block face has

any deformity to it caused by a funky mold - just smear a thin layer of
OCT 
over block face to fill in these depressions, etc.  to have a flatter
block 
face before trimming.   Thin layer is important as we tried to avoid any

excess warming of block once it is frozen solid.

Have fun!!


At 07:07 PM 1/25/2005, you wrote:
>Hi,
>         I'm snap freezing kidney biopsies in OCT using isopentane that
is 
> chilled with liquid nitrogen. I'm having a problem where the area that

> the biopsy is gets raised up above the surface of the block. I'm not
sure 
> what is going wrong and more importantly what to do about it. Has
anyone 
> had similar problems and how did you solve it?
>
>Thanks,
>Amos Brooks
>
>
>
>_______________________________________________
>Histonet mailing list
>Histonet@lists.utsouthwestern.edu
>http://lists.utsouthwestern.edu/mailman/listinfo/histonet

Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)





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

Message: 21
Date: Wed, 26 Jan 2005 09:33:09 -0700
From: Gayle Callis 
Subject: Re: [Histonet] RE: Fluorescence staining
To: "C.M. van der Loos" ,
	Histonet@lists.utsouthwestern.edu
Message-ID:
	<6.0.0.22.1.20050126092847.01b583c0@gemini.msu.montana.edu>
Content-Type: text/plain; charset="us-ascii"; format=flowed

Chris and Eva,

Vectashield also come in a Hard Set, so you get permanent coverslipping
(no 
coverglass sliding around!) as does the Molecular Probes Prolong Gold
ready 
to use.

If you want a counterfluorescence marker for nuclei, and have the 
appropriate DAPI filter on microscope, you can buy these with DAPI which

"counterstains via fluorescence" the DNA in cells.


At 12:52 AM 1/26/2005, you wrote:

>    Dear Eva,
>
>    To my knowledge most of the traditional fluorochromes like FITC,
>    rhodamine, doesn't stand dehydration at all. So in your case it
fully
>    depends on what type of fluorochrome you end up with after the TSA
>    procedure.
>
>    If you end up with FITC, the use of VectaShield
>    ([1]www.vectorlabs.com) for mounting is highly recommended for
>    the prevention of photobleaching.
>
>    Chris van der Loos, PhD
>    Dept. of Pathology
>    Academical Medical Center M2-230
>    Meibergdreef 9
>    NL-1105 AZ Amsterdam
>    The Netherlands
>
>    phone:  +31 20 5665631
>    fax:    +31 20 6960389
>    e-mail: [2]c.m.vanderloos@amc.uva.nl

Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)





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

Message: 22
Date: Wed, 26 Jan 2005 10:33:22 -0600
From: ajennings@unmc.edu
Subject: Re: [Histonet] Freezing Biopsies
Cc: histonet@lists.utsouthwestern.edu,
	histonet-bounces@lists.utsouthwestern.edu,	Amos Brooks
	
Message-ID:
	

Content-Type: text/plain; charset=US-ASCII





Bernice from Instrumedics I am sure can give you better insight into
what
use to be called "Gentle Jane" freezing. Helps with heat extraction and
overall consistent freezing.
It may help.

I use to have the same problem with whole mouse embryos, because they
were
freezing from the outside in and by the time the liver froze/expanded it
would pop out of the block. I know biopsies are by far smaller but it
could
be the same general theory with the OCT freezing from the outside around
the sample and the sample having no place else to expand. It actually
helped me to freeze in the lN2 vapor instead of dropping the sample into
isopentane. The time difference was negligible and I didn't get that pop
out peak that I believe you are referring to. The freezing occurred from
the bottom of the boat to the top instead from all sides at once. It
might
not be appropriate for all but it has worked in my lab for many many
years.

just sharing a thought


On Jan 25, 2005, at 8:07 PM, Amos Brooks wrote:

> Hi,
>            I'm snap freezing kidney biopsies in OCT using isopentane
that
is
> chilled with liquid nitrogen. I'm having a problem where the area that
> the biopsy is gets raised up above the surface of the block. I'm not
> sure what is going wrong and more importantly what to do about it. Has
> anyone had similar problems and how did you solve it?
>
> Thanks,
> Amos Brooks




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

Message: 23
Date: Wed, 26 Jan 2005 09:36:26 -0700
From: "Elizabeth Chlipala" 
Subject: RE: [Histonet] Manual fixation/processing of reconstructed
	skincultures grown on polycarbonate filters
To: "'Colin Smith'" ,
	
Message-ID: <000001c503c5$2e345770$76d48a80@AMY>
Content-Type: text/plain;	charset="us-ascii"

Colin

We have worked quite a bit with the epioral and epigingival cultures
from Mattek.  We have stained with H&E, Ki-67 and Cleaved caspase 3. We
processed these specimens on a tissue processor, but you will be able to
do this by hand.  I'm not sure of the size of your culture wells, but we
used an 8mm biopsy punch to remove the constructs from the wells,
processed the construct whole between two biopsy pads and bisected with
a razor blade prior to embedding on end.  As far as fixation 10% NBF
will work.  The wells were labeled on the side and the entire well was
placed in a 50 ml conical tube with fixative.  After adequate fixation
the specimens were grossed, processed and embedded into paraffin.  We
processed during the day (not overnight) at 15 minutes per station.  We
sectioned at 4 microns and stained with both H&E and various
Immunohistochemical stains, I can send you images if you are interested.
If you need more info, give me a call or e-mail me back.  Also we are
writing an article that will be in the spring edition of Histologic that
basically covers the methods (both processing and analysis) we used to
assess proliferation in these in vitro tissue models.  

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
P.O. Box 18592
Boulder, Colorado 80308
Office: (303) 735-5001
Fax: (303) 735-3540
liz@premierlab.com
www.premierlab.com
 
Ship to Address:
Premier Laboratory
University of Colorado
MCDB, Room A3B40
Boulder, Colorado 80309

-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu
[mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of Colin
Smith
Sent: Wednesday, January 26, 2005 8:07 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Manual fixation/processing of reconstructed
skincultures grown on polycarbonate filters

Can anyone help?
I am currently setting up a basic histology system for analysing
reconstructed tissues grown in culture on polycarbonate filters, similar
to
Mattek's Epiderm. We currently have begged borrowed or stolen,
equipment-wise:
two water baths (ambient - 100C), a circulating warm air oven, a rocking
microtome and cold blocks, along with glass staining jars/racks, forceps
etc.
At the moment, equipment such as a wax dispenser, purpose built cooling
plates etc are out of the question until I can show that we can show
that the
model we are growing is validated, which will include
immunohistochemistry of
cytokeratin and cell adhesion markers etc. Thus we are in the catch 22
situation of having to prove something without the equipment needed to
do the
job, in order to justify buying the equipment!! But thats accountants
for
you, go figure.
Anyway, can anyone give any useful pointers/protocols for
fixing/processing/embedding/sectioning such tissues using such primative
equipment
Any help, no matter how trivial will be greatfully received.
 
Many Thanks in Advance,
 
Colin Smith BSc CBiol MIBiol Eurotox Registered Toxicologist
In Vitro Toxicologist 
Thor Specialities UK Ltd
Wincham Avenue,
Northwich
CW9 6GB
 
Tel: +44 1606 818873
Fax: +44 1606 818801
colins@thor.uk.com
 
 

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

Message: 24
Date: Wed, 26 Jan 2005 09:48:38 -0800 (PST)
From: Jarrin Miguel 
Subject: [Histonet] mouse eye lens
To: histonet@lists.utsouthwestern.edu
Message-ID: <20050126174838.64967.qmail@web41901.mail.yahoo.com>
Content-Type: text/plain; charset=us-ascii

Dear all,
 
Let me introduce myself. My name is Miguel and I am PhD student. I am
considering to carry out immunochemistry and in situ hybridization
studies in parafin sections of eye lens in adult mouse. I would like
receive futher information about the best fixative that use. I have
certain experience with Paraformaldehide, but I have been advised about
Davison's fixative and Penfix. I never have heared about Penfix before.
Somebody has protocols or receipt for the penfix?
 
Thanking in advance for your help
 
Miguel   

		
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