[Histonet] RE: Histonet Digest, Vol 64, Issue 7

[Valantou Grover]

Hartmann's fixative: 
Hello All, 

I first became aware of this fixative in when still doing my internship from
histology school at Compunet Clinical laboratory in Dayton, OH.  I was asked
by my technical director, Alice L. Trent to make it up from scratch, even
through it is called Hartmann's Fixitive it is Davidson's modification.  I
also learned about it when studying intestinal biopsies and colon resections
at another OSH in Toledo in 1997.  Not only does tissue especially some of
the fat surrounding the colon looked amazingly better than with 10% NBF, it
lakes RBCs, penetrates pretty rapidly, and causes much less tissue shrinkage
than 10% NBF or the mercuric fixatives (such as B-5). It is also better
showing tumor patterns :nodularity in  lymphoma, stromal patterns in
soft-tissue and mucinous lesions. It was referred to as lymph node revealing
solution because, it causes DNA-rich tissue to differentially turn white
quickly.  Colon malignancies fixed in Hartmann's fixative become  visually
as well as touch sensitive when you smash the fat you're your finger to
discover smaller lymph nodes (grossly...this is a sub-grossing technique)
when concerning the depth of penetration and a "deepest-penetrated" block
selected. This is a valuable tool in node dissections, where 1-3 mm nodes
can be found by smashing the fat with your fingers even while wearing
gloves.  The nodes become palpably distinct from fat and turn white (lymph
node revealing solution) and as seen in a cross section on an H and E slide)
We also used it a couple of times at PH for a colon CA case for one of the
chief residents at the time asked me a question if I have ever heard about
this fixative and he needed it to find the rest of the lymph nodes in one of
his colon CA for the 1:00 pm daily conference.  He has also tried it for
multifocal cancer in a mastectomy case, because of some literature he
followed up on from my article that was saved from my early histology
education. Another formulation includes diethyl ether is used for axillary
lymph node dissections for breast cancer.  Small bronchi richly surrounded
by chronic inflammatory cells are white on cross-section. Hartmann's is a
gentler fixative, than the B-5 containing fixatives as well as 10% formalin.

  .

Formula:

10% NBF -  24.3%
95% ETOH -32.4%
acetic acid, glacial tech grade- 10.9%                      
deionized water-32.4%

of a solution totaling 100% you can adjust the volume of liquid to make
100%, so you can make 100 ml or 20 liters using these ratios.

 

References:

Loren R, et. al., "Lymph node revealing solution: A new method for detection
of minute axillary lymph nodes in breast cancer specimens." Am J Surg
Pathol. 1997; 21(11):1387-1390. ( this is the article given to me by my
technical director Alice Louise Trent MLT(ASCP), HT(ASCP) at Compunet
Clinical Labs in Dayton Ohio during my Histology School internship) 

Many thanks Alice!!!!!!

Sincerely, 



Valantou Grover, HT/HTL(ASCP), PA, MBA
Biosciences Product Line Manager 
Polysciences, Inc. 
400 Valley Road 
Warrington, PA 18976
Fax: 1-800-323-3291
Phone number: 1-800-523-2575 X7418
Direct:1-215-488-7418
Cell phone: 1-215-409-8327
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Subject: Histonet Digest, Vol 64, Issue 7

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Today's Topics:

   1. staining for elastic fiber of artery in rat lung  (Amy Lee)
   2. vector blue- xylene soluble- any alternatives (anjan kumar)
   3. (no subject) (Matthew Bingham)
   4. problem eyes (Perry, Margaret)
   5. Re: problem eyes (Rene J Buesa)
   6. Re: problem eyes (Va Paula Sicurello)
   7. Re: problem eyes (Rene J Buesa)
   8. Re: problem eyes (Merced Leiker)
   9. Water bubble on slides (Vanessa J. Phelan)
  10. RE: problem eyes (Connolly, Brett M)
  11. RE: vector blue- xylene soluble- any alternatives (Patsy Ruegg)
  12. Re: Water bubble on slides (Merced Leiker)
  13. Autotechnicon model 2A (Va Paula Sicurello)
  14. RE: problem eyes (Tony Henwood)
  15. colon cancer lymph nodes (Steve Eagle)
  16. AXOn staining (TF)
  17. Natural Killer Cell antibody (Kim O'Sullivan)
  18. Re: problem eyes (Anne van Binsbergen)
  19. RE: We are buying a new Tissue Processor and need imput
      (Walzer Susan)
  20. staining brain vessels (iskaliora)
  21. RE: Natural Killer Cell antibody (Pritchard, Michele)
  22. RE: problem eyes (Bernice Frederick)
  23. Re: staining brain vessels (TF)


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

Message: 1
Date: Wed, 4 Mar 2009 10:21:43 -0800 (PST)
From: Amy Lee <amylee779 <@t> yahoo.com>
Subject: [Histonet] staining for elastic fiber of artery in rat lung 
To: histonet <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <937896.88292.qm <@t> web38006.mail.mud.yahoo.com>
Content-Type: text/plain; charset=iso-8859-1

Hello histonetters,
 
Would you teach me what is best stain for elastic fiber in rat lung except
weigert's stain?
 
I want to look at arteries.
 
Thanks in advance,
Weihua


      

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

Message: 2
Date: Thu, 5 Mar 2009 00:34:32 +0530
From: anjan kumar <drvet_anjan <@t> hotmail.com>
Subject: [Histonet] vector blue- xylene soluble- any alternatives
To: <histonet-requests <@t> lists.utsouthwestern.edu>, triple
	immunohistochem	<histonet <@t> lists.utsouthwestern.edu>
Message-ID: <BLU149-W4DED6BF75DCD11FC158C78BA70 <@t> phx.gbl>
Content-Type: text/plain; charset="iso-8859-1"


hello everyone,

                    i have a problem which i dont know how to tackle, the
problem is that i have purchased vector blue as a chromogen and it is xylene
soluble can anyone suggest me how to avoid this.... as funds are low i cant
purchase xylene substitutes. 

 

second question been, ...sorry this has been a addition ......

what is the difference between the DPX mountant and other permanent mounting
media like vectamount. kindly answer these questions...

 

regards,

Anjan Kumar

Junior research scientist

Veterinary Pathology

Madras veterinary college.

_________________________________________________________________
Ready for MARRIAGE? Join MSN Matrimony!
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------------------------------

Message: 3
Date: Wed, 4 Mar 2009 11:43:28 -0800 (PST)
From: Matthew Bingham <binghammg <@t> yahoo.com>
Subject: [Histonet] (no subject)
To: histonet <@t> lists.utsouthwestern.edu
Message-ID: <322914.55412.qm <@t> web63507.mail.re1.yahoo.com>
Content-Type: text/plain; charset=us-ascii

As many of you may or may not know, the Histobath countertop freezing bath
has
been discontinued by Thermo (formerly known as Thermo-Shandon,
Shandon-Lipshaw,
etc.). I was told that the freon that is used to allow the unit to cool to
-60
is no longer allowed by the EPA to be used in ultra low refrigeration units.
I
know there is a floor model, the Clini-RF, at brightinstruments.com which
uses a
two types of freon in isolated side by side systems, but it is a rather
large
unit (20"D x 18"W x 40"H). Our frozen section room, as are most, is a small
room with very limited space. Therefore, have any of you found a solution
for a
countertop model (i.e. 19"D x 9"W x 15"H)freezing bath to be used in frozen
section rooms? I appreciate the help.

Thanks,

Matthew Bingham, PA(ASCP)CMAnatomic Pathology Supervisor
Phoenix Children's Hospital
Department of Pathology
1919 E. Thomas Road
Phoenix, AZ 85016
phone: 602-546-1318
fax: 602-546-1284
email: mbingham <@t> phoenixchildrens.com



      

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

Message: 4
Date: Wed, 4 Mar 2009 15:04:23 -0600
From: "Perry, Margaret" <Margaret.Perry <@t> sdstate.edu>
Subject: [Histonet] problem eyes
To: "histonet <@t> lists.utsouthwestern.edu"
	<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
	<FCA5EF47F9BC694CBB4C58FEA042196343A55195C9 <@t> SDSU-MBX.jacks.local>
Content-Type: text/plain; charset="us-ascii"

Please help!  We have been trying to cut whole eyes from a pig.  They need
to be nice enough for a publication.  We are having the devil of a time
because no matter what we do there are wrinkles.  If we turn the waterbath
up to stretch things out the retina detaches.  Do any of you have
suggestions?  We uses Surgipath  EM400 for embedding paraffin and and their
infiltration medium.  Is this to soft? The lowest we can turn our waterbath
is 38 degrees C.

Margaret Perry HT (ASCP)
IHC Lab Manager Veterinary Science
Animal Disease Research and Diagnostic Lab
South Dakota State University
Box 2175 North Campus Drive
Brookings SD 57007



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

Message: 5
Date: Wed, 4 Mar 2009 13:14:56 -0800 (PST)
From: Rene J Buesa <rjbuesa <@t> yahoo.com>
Subject: Re: [Histonet] problem eyes
To: "histonet <@t> lists.utsouthwestern.edu"
	<histonet <@t> lists.utsouthwestern.edu>,	"Perry, Margaret"
	<Margaret.Perry <@t> sdstate.edu>
Message-ID: <240791.54814.qm <@t> web65712.mail.ac4.yahoo.com>
Content-Type: text/plain; charset=iso-8859-1

Add a few drops (4-5) of liquid detergent, but not dishwasher detergent
(because it will dissolve the paraffin).
reni J.

--- On Wed, 3/4/09, Perry, Margaret <Margaret.Perry <@t> sdstate.edu> wrote:

From: Perry, Margaret <Margaret.Perry <@t> sdstate.edu>
Subject: [Histonet] problem eyes
To: "histonet <@t> lists.utsouthwestern.edu" <histonet <@t> lists.utsouthwestern.edu>
Date: Wednesday, March 4, 2009, 4:04 PM

Please help!  We have been trying to cut whole eyes from a pig.  They need
to be
nice enough for a publication.  We are having the devil of a time because no
matter what we do there are wrinkles.  If we turn the waterbath up to
stretch
things out the retina detaches.  Do any of you have suggestions?  We uses
Surgipath  EM400 for embedding paraffin and and their infiltration medium.
Is
this to soft? The lowest we can turn our waterbath is 38 degrees C.

Margaret Perry HT (ASCP)
IHC Lab Manager Veterinary Science
Animal Disease Research and Diagnostic Lab
South Dakota State University
Box 2175 North Campus Drive
Brookings SD 57007

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



      

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

Message: 6
Date: Wed, 4 Mar 2009 13:23:18 -0800 (PST)
From: Va Paula Sicurello <vapatpxs <@t> yahoo.com>
Subject: Re: [Histonet] problem eyes
To: "histonet <@t> lists.utsouthwestern.edu"
	<histonet <@t> lists.utsouthwestern.edu>,	MargaretPerry
	<Margaret.Perry <@t> sdstate.edu>, rjbuesa <@t> yahoo.com
Message-ID: <787849.53788.qm <@t> web46101.mail.sp1.yahoo.com>
Content-Type: text/plain; charset=iso-8859-1


Really?  Liquid laundry soap?  Does this help with all wrinkles in large
specimens?


Paula Sicurello
VA Medical Center San Diego
Veterans Medical Research Foundation (VMRF) 
Core Microscope Facility, room B141
3350 La Jolla Village Dr., MC151
San Diego, CA 92161
858-552-8585 x2397


--- On Wed, 3/4/09, Rene J Buesa <rjbuesa <@t> yahoo.com> wrote:

> From: Rene J Buesa <rjbuesa <@t> yahoo.com>
> Subject: Re: [Histonet] problem eyes
> To: "histonet <@t> lists.utsouthwestern.edu"
<histonet <@t> lists.utsouthwestern.edu>, "Perry, Margaret"
<Margaret.Perry <@t> sdstate.edu>
> Date: Wednesday, March 4, 2009, 9:14 PM
> Add a few drops (4-5) of liquid
> detergent, but not dishwasher detergent (because it will
> dissolve the paraffin).
> reni J.
> 
> --- On Wed, 3/4/09, Perry, Margaret <Margaret.Perry <@t> sdstate.edu>
> wrote:
> 
> From: Perry, Margaret <Margaret.Perry <@t> sdstate.edu>
> Subject: [Histonet] problem eyes
> To: "histonet <@t> lists.utsouthwestern.edu"
> <histonet <@t> lists.utsouthwestern.edu>
> Date: Wednesday, March 4, 2009, 4:04 PM
> 
> Please help!  We have been trying to cut whole eyes
> from a pig.  They need to be
> nice enough for a publication.  We are having the
> devil of a time because no
> matter what we do there are wrinkles.  If we turn the
> waterbath up to stretch
> things out the retina detaches.  Do any of you have
> suggestions?  We uses
> Surgipath  EM400 for embedding paraffin and and their
> infiltration medium.  Is
> this to soft? The lowest we can turn our waterbath is 38
> degrees C.
> 
> Margaret Perry HT (ASCP)
> IHC Lab Manager Veterinary Science
> Animal Disease Research and Diagnostic Lab
> South Dakota State University
> Box 2175 North Campus Drive
> Brookings SD 57007
> 
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
> 
> 
> 
> 
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
> 


      




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

Message: 7
Date: Wed, 4 Mar 2009 13:27:32 -0800 (PST)
From: Rene J Buesa <rjbuesa <@t> yahoo.com>
Subject: Re: [Histonet] problem eyes
To: "histonet <@t> lists.utsouthwestern.edu"
	<histonet <@t> lists.utsouthwestern.edu>,	MargaretPerry
	<Margaret.Perry <@t> sdstate.edu>,	Va Paula Sicurello
<vapatpxs <@t> yahoo.com>
Message-ID: <644825.64523.qm <@t> web65712.mail.ac4.yahoo.com>
Content-Type: text/plain; charset=iso-8859-1

Yes, really with any type of large (and small) sections. The liquid soap
will reduce the surface tension of water (more than heat) allowing the
sections to expand easily. Try it..
Reni J.

--- On Wed, 3/4/09, Va Paula Sicurello <vapatpxs <@t> yahoo.com> wrote:

From: Va Paula Sicurello <vapatpxs <@t> yahoo.com>
Subject: Re: [Histonet] problem eyes
To: "histonet <@t> lists.utsouthwestern.edu" <histonet <@t> lists.utsouthwestern.edu>,
"MargaretPerry" <Margaret.Perry <@t> sdstate.edu>, rjbuesa <@t> yahoo.com
Date: Wednesday, March 4, 2009, 4:23 PM

Really?  Liquid laundry soap?  Does this help with all wrinkles in large
specimens?


Paula Sicurello
VA Medical Center San Diego
Veterans Medical Research Foundation (VMRF) 
Core Microscope Facility, room B141
3350 La Jolla Village Dr., MC151
San Diego, CA 92161
858-552-8585 x2397


--- On Wed, 3/4/09, Rene J Buesa <rjbuesa <@t> yahoo.com> wrote:

> From: Rene J Buesa <rjbuesa <@t> yahoo.com>
> Subject: Re: [Histonet] problem eyes
> To: "histonet <@t> lists.utsouthwestern.edu"
<histonet <@t> lists.utsouthwestern.edu>, "Perry, Margaret"
<Margaret.Perry <@t> sdstate.edu>
> Date: Wednesday, March 4, 2009, 9:14 PM
> Add a few drops (4-5) of liquid
> detergent, but not dishwasher detergent (because it will
> dissolve the paraffin).
> reni J.
> 
> --- On Wed, 3/4/09, Perry, Margaret <Margaret.Perry <@t> sdstate.edu>
> wrote:
> 
> From: Perry, Margaret <Margaret.Perry <@t> sdstate.edu>
> Subject: [Histonet] problem eyes
> To: "histonet <@t> lists.utsouthwestern.edu"
> <histonet <@t> lists.utsouthwestern.edu>
> Date: Wednesday, March 4, 2009, 4:04 PM
> 
> Please help!  We have been trying to cut whole eyes
> from a pig.  They need to be
> nice enough for a publication.  We are having the
> devil of a time because no
> matter what we do there are wrinkles.  If we turn the
> waterbath up to stretch
> things out the retina detaches.  Do any of you have
> suggestions?  We uses
> Surgipath  EM400 for embedding paraffin and and their
> infiltration medium.  Is
> this to soft? The lowest we can turn our waterbath is 38
> degrees C.
> 
> Margaret Perry HT (ASCP)
> IHC Lab Manager Veterinary Science
> Animal Disease Research and Diagnostic Lab
> South Dakota State University
> Box 2175 North Campus Drive
> Brookings SD 57007
> 
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
> 
> 
> 
> 
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
> 







      

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

Message: 8
Date: Wed, 04 Mar 2009 16:29:12 -0500
From: Merced Leiker <leiker <@t> buffalo.edu>
Subject: Re: [Histonet] problem eyes
To: histonet <@t> lists.utsouthwestern.edu
Message-ID: <7FD71F250513F2B17D70DB05 <@t> bchwxp2702.ad.med.buffalo.edu>
Content-Type: text/plain; charset=iso-8859-1; format=flowed

Good question! I'd like to know that, too! But with any size specimen, 
please!

--On Wednesday, March 04, 2009 1:23 PM -0800 Va Paula Sicurello 
<vapatpxs <@t> yahoo.com> wrote:

>
> Really?  Liquid laundry soap?  Does this help with all wrinkles in large
> specimens?
>
>
> Paula Sicurello
> VA Medical Center San Diego
> Veterans Medical Research Foundation (VMRF)
> Core Microscope Facility, room B141
> 3350 La Jolla Village Dr., MC151
> San Diego, CA 92161
> 858-552-8585 x2397
>
>
> --- On Wed, 3/4/09, Rene J Buesa <rjbuesa <@t> yahoo.com> wrote:
>
>> From: Rene J Buesa <rjbuesa <@t> yahoo.com>
>> Subject: Re: [Histonet] problem eyes
>> To: "histonet <@t> lists.utsouthwestern.edu"
>> <histonet <@t> lists.utsouthwestern.edu>, "Perry, Margaret"
>> <Margaret.Perry <@t> sdstate.edu> Date: Wednesday, March 4, 2009, 9:14 PM
>> Add a few drops (4-5) of liquid
>> detergent, but not dishwasher detergent (because it will
>> dissolve the paraffin).
>> reni J.
>>
>> --- On Wed, 3/4/09, Perry, Margaret <Margaret.Perry <@t> sdstate.edu>
>> wrote:
>>
>> From: Perry, Margaret <Margaret.Perry <@t> sdstate.edu>
>> Subject: [Histonet] problem eyes
>> To: "histonet <@t> lists.utsouthwestern.edu"
>> <histonet <@t> lists.utsouthwestern.edu>
>> Date: Wednesday, March 4, 2009, 4:04 PM
>>
>> Please help!  We have been trying to cut whole eyes
>> from a pig.  They need to be
>> nice enough for a publication.  We are having the
>> devil of a time because no
>> matter what we do there are wrinkles.  If we turn the
>> waterbath up to stretch
>> things out the retina detaches.  Do any of you have
>> suggestions?  We uses
>> Surgipath  EM400 for embedding paraffin and and their
>> infiltration medium.  Is
>> this to soft? The lowest we can turn our waterbath is 38
>> degrees C.
>>
>> Margaret Perry HT (ASCP)
>> IHC Lab Manager Veterinary Science
>> Animal Disease Research and Diagnostic Lab
>> South Dakota State University
>> Box 2175 North Campus Drive
>> Brookings SD 57007
>>
>> _______________________________________________
>> Histonet mailing list
>> Histonet <@t> lists.utsouthwestern.edu
>> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>>
>>
>>
>>
>> _______________________________________________
>> Histonet mailing list
>> Histonet <@t> lists.utsouthwestern.edu
>> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>>
>
>
>
>
>
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>



Merced M Leiker
Research Technician II
354 Biomedical Research Building
School of Medicine and Biomedical Sciences
State University of New York at Buffalo
3435 Main St, Buffalo, NY 14214
Ph: (716) 829-6033
Fx: (716) 829-2725

No trees were harmed in the sending of this email.
However, many electrons were severely inconvenienced.




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

Message: 9
Date: Wed, 4 Mar 2009 16:35:14 -0500
From: "Vanessa J. Phelan" <vjp2105 <@t> columbia.edu>
Subject: [Histonet] Water bubble on slides
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <C5D45E42.1AD%vjp2105 <@t> columbia.edu>
Content-Type: text/plain; charset="US-ASCII"

Hi everyone,

Quick question, I am finding after cutting a lot of slides for H&Es onto VWR
Superfrost Plus slides (these are the only slides we have at the moment)
that when I lift sections from the water bath ( of distilled water) a water
bubble stays under the sections on the slide. When I lie them flat on the
hot plate to dry the water bubble is tending to distort the tissue by
escaping out through he tissue, eventually. I have not experienced this
before, the water has ran off the slide no problem. Would it be the slides
that are the problem or anything I might need to add to the water?

I have just ordered a histology oven, this may be the solution...drying the
slides standing up!

Vanessa




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

Message: 10
Date: Wed, 4 Mar 2009 16:36:19 -0500
From: "Connolly, Brett M" <brett_connolly <@t> merck.com>
Subject: RE: [Histonet] problem eyes
To: "Perry, Margaret" <Margaret.Perry <@t> sdstate.edu>,
	<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
	<63EA0607835FBA4689CEA9EA8B48269201C38A0B <@t> usctmx1141.merck.com>
Content-Type: text/plain; charset="us-ascii"

My suggestion would be to follow the procedure of the old ocular guru
from the AFIP, Peter Emanuale. He taught me how the section human and
rabbit eyes. It involves using 2 water baths, one of which is at room
temp... and you should not use charged/coated slides.

Check our his chapter on ocular histotechnology in the 1992 edition of
the AFIP Lab Methods manual (it has step by step illustrations)

Brett M. Connolly, Ph.D.
Research Fellow, Imaging Research
Merck & Co., Inc.
PO Box 4, WP-44K
West Point, PA 19486
PH 215-652-2501 fax. 215-993-6803
e-mail. brett_connolly <@t> merck.com
   

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Perry,
Margaret
Sent: Wednesday, March 04, 2009 4:04 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] problem eyes

Please help!  We have been trying to cut whole eyes from a pig.  They
need to be nice enough for a publication.  We are having the devil of a
time because no matter what we do there are wrinkles.  If we turn the
waterbath up to stretch things out the retina detaches.  Do any of you
have suggestions?  We uses Surgipath  EM400 for embedding paraffin and
and their infiltration medium.  Is this to soft? The lowest we can turn
our waterbath is 38 degrees C.

Margaret Perry HT (ASCP)
IHC Lab Manager Veterinary Science
Animal Disease Research and Diagnostic Lab
South Dakota State University
Box 2175 North Campus Drive
Brookings SD 57007

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

Message: 11
Date: Wed, 4 Mar 2009 14:38:57 -0700
From: "Patsy Ruegg" <pruegg <@t> ihctech.net>
Subject: RE: [Histonet] vector blue- xylene soluble- any alternatives
To: "'anjan kumar'" <drvet_anjan <@t> hotmail.com>,
	<histonet-requests <@t> lists.utsouthwestern.edu>,	"'triple
	immunohistochem'" <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <011DFB4829B140CE8DF5E5222EAE7497 <@t> prueggihctechlt>
Content-Type: text/plain;	charset="us-ascii"

If I am not mistaken vectamount is an aqueous mountant, dpx is
xylene/toluene based and requires going thru alcohols and xylene to mount.
One thing I do and you can try it, is airdry the slides and the use
permanent mounting media without going thru alcohols and xylene, keep in
mind there is a little solvent in the media so it may have an effect.  I do
this with Fast Red substrate chromogen for AP have not tried it on vector
blue.  The other option is to use an aqueous mountant and seal the coverslip
edges with clear nail polish.
Patsy

Patsy Ruegg, HT(ASCP)QIHC
IHCtech
12635 Montview Blvd. Ste.215
Aurora, CO 80045
720-859-4060
fax 720-859-4110
www.ihctech.net 
www.ihcrg.org

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of anjan kumar
Sent: Wednesday, March 04, 2009 12:05 PM
To: histonet-requests <@t> lists.utsouthwestern.edu; triple immunohistochem
Subject: [Histonet] vector blue- xylene soluble- any alternatives


hello everyone,

                    i have a problem which i dont know how to tackle, the
problem is that i have purchased vector blue as a chromogen and it is xylene
soluble can anyone suggest me how to avoid this.... as funds are low i cant
purchase xylene substitutes. 

 

second question been, ...sorry this has been a addition ......

what is the difference between the DPX mountant and other permanent mounting
media like vectamount. kindly answer these questions...

 

regards,

Anjan Kumar

Junior research scientist

Veterinary Pathology

Madras veterinary college.

_________________________________________________________________
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------------------------------

Message: 12
Date: Wed, 04 Mar 2009 16:50:29 -0500
From: Merced Leiker <leiker <@t> buffalo.edu>
Subject: Re: [Histonet] Water bubble on slides
To: "Vanessa J. Phelan" <vjp2105 <@t> columbia.edu>,
	histonet <@t> lists.utsouthwestern.edu
Message-ID: <CB4313477EEE52456DB3850A <@t> bchwxp2702.ad.med.buffalo.edu>
Content-Type: text/plain; charset=us-ascii; format=flowed

Hi Vanessa,

I have read in at least one online protocol and have also been advised by 
our excellent and experienced (former) histotech who used to be here at UB 
NOT to warm the slides without air-drying first.  We typically air-dry the 
slides, yes, standing up, overnight, before warming them.

Merced

--On Wednesday, March 04, 2009 4:35 PM -0500 "Vanessa J. Phelan" 
<vjp2105 <@t> columbia.edu> wrote:

> Hi everyone,
>
> Quick question, I am finding after cutting a lot of slides for H&Es onto
> VWR Superfrost Plus slides (these are the only slides we have at the
> moment) that when I lift sections from the water bath ( of distilled
> water) a water bubble stays under the sections on the slide. When I lie
> them flat on the hot plate to dry the water bubble is tending to distort
> the tissue by escaping out through he tissue, eventually. I have not
> experienced this before, the water has ran off the slide no problem.
> Would it be the slides that are the problem or anything I might need to
> add to the water?
>
> I have just ordered a histology oven, this may be the solution...drying
> the slides standing up!
>
> Vanessa
>
>
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>



Merced M Leiker
Research Technician II
354 Biomedical Research Building
School of Medicine and Biomedical Sciences
State University of New York at Buffalo
3435 Main St, Buffalo, NY 14214
Ph: (716) 829-6033
Fx: (716) 829-2725

No trees were harmed in the sending of this email.
However, many electrons were severely inconvenienced.




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

Message: 13
Date: Wed, 4 Mar 2009 14:39:33 -0800 (PST)
From: Va Paula Sicurello <vapatpxs <@t> yahoo.com>
Subject: [Histonet] Autotechnicon model 2A
To: HistoNet <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <969177.76764.qm <@t> web46110.mail.sp1.yahoo.com>
Content-Type: text/plain; charset=us-ascii


Hi All,

Does anybody have an owner's manual to an Autotechnicon 2A?  I've inherited
this 42 year old beasty and can't remember how to work the delay timer.

Yes, I am old enough to have used one of these R2D2 units in the past.

If you can help me, I'd be much obliged.

Paula  :-)

Paula Sicurello
VA Medical Center San Diego
Veterans Medical Research Foundation (VMRF) 
Core Microscope Facility, room B141
3350 La Jolla Village Dr., MC151
San Diego, CA 92161
858-552-8585 x2397


      




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

Message: 14
Date: Thu, 5 Mar 2009 09:41:39 +1100
From: "Tony Henwood" <AnthonyH <@t> chw.edu.au>
Subject: RE: [Histonet] problem eyes
To: "Va Paula Sicurello" <vapatpxs <@t> yahoo.com>,
	<histonet <@t> lists.utsouthwestern.edu>, "MargaretPerry"
	<Margaret.Perry <@t> sdstate.edu>, <rjbuesa <@t> yahoo.com>
Message-ID: <B9EAF61856077F47BF9BE2F89AFC555202FB0246 <@t> hedwig.nch.kids>
Content-Type: text/plain; charset="iso-8859-1"

Doesn't help with the wrinkles in OLD large Histotechs.

Sorry, couldn't resist

Regards

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager & Senior Scientist
Tel: 612 9845 3306
Fax: 612 9845 3318
the children's hospital at westmead 
Cnr Hawkesbury Road and Hainsworth Street, Westmead 
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 




-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Va Paula
Sicurello
Sent: Thursday, 5 March 2009 8:23 AM
To: histonet <@t> lists.utsouthwestern.edu; MargaretPerry; rjbuesa <@t> yahoo.com
Subject: Re: [Histonet] problem eyes



Really?  Liquid laundry soap?  Does this help with all wrinkles in large
specimens?


Paula Sicurello
VA Medical Center San Diego
Veterans Medical Research Foundation (VMRF) 
Core Microscope Facility, room B141
3350 La Jolla Village Dr., MC151
San Diego, CA 92161
858-552-8585 x2397


--- On Wed, 3/4/09, Rene J Buesa <rjbuesa <@t> yahoo.com> wrote:

> From: Rene J Buesa <rjbuesa <@t> yahoo.com>
> Subject: Re: [Histonet] problem eyes
> To: "histonet <@t> lists.utsouthwestern.edu" 
> <histonet <@t> lists.utsouthwestern.edu>, "Perry, Margaret"
<Margaret.Perry <@t> sdstate.edu>
> Date: Wednesday, March 4, 2009, 9:14 PM
> Add a few drops (4-5) of liquid
> detergent, but not dishwasher detergent (because it will
> dissolve the paraffin).
> reni J.
> 
> --- On Wed, 3/4/09, Perry, Margaret <Margaret.Perry <@t> sdstate.edu>
> wrote:
> 
> From: Perry, Margaret <Margaret.Perry <@t> sdstate.edu>
> Subject: [Histonet] problem eyes
> To: "histonet <@t> lists.utsouthwestern.edu"
> <histonet <@t> lists.utsouthwestern.edu>
> Date: Wednesday, March 4, 2009, 4:04 PM
> 
> Please help!  We have been trying to cut whole eyes
> from a pig.  They need to be
> nice enough for a publication.  We are having the
> devil of a time because no
> matter what we do there are wrinkles.  If we turn the waterbath up to 
> stretch things out the retina detaches.  Do any of you have
> suggestions?  We uses
> Surgipath  EM400 for embedding paraffin and and their
> infiltration medium.  Is
> this to soft? The lowest we can turn our waterbath is 38
> degrees C.
> 
> Margaret Perry HT (ASCP)
> IHC Lab Manager Veterinary Science
> Animal Disease Research and Diagnostic Lab
> South Dakota State University
> Box 2175 North Campus Drive
> Brookings SD 57007
> 
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu 
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
> 
> 
> 
> 
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu 
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
> 


      


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

Message: 15
Date: Wed, 4 Mar 2009 18:34:38 -0800 (PST)
From: Steve Eagle <stevegeagle <@t> yahoo.com>
Subject: [Histonet] colon cancer lymph nodes
To: histonet <@t> lists.utsouthwestern.edu
Message-ID: <820129.73519.qm <@t> web36201.mail.mud.yahoo.com>
Content-Type: text/plain; charset=us-ascii


An additional step that often is helpful is to submit pericolonic adipose
tissue with vessels... frequently you can find microscopic lymph
nodes/lymphoid aggregates.


      



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

Message: 16
Date: Thu, 5 Mar 2009 10:42:45 +0800
From: "TF" <tifei <@t> foxmail.com>
Subject: [Histonet] AXOn staining
To: "nancy.walker" <nancy.walker <@t> sanofi-synthelabo.com>
Cc: histonet <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <200903051042396493204 <@t> foxmail.com>
Content-Type: text/plain;	charset="us-ascii"

Re: 
Hi Nancy, do Tuj1 (beta 3 tubilin) stain nicely in adult rat brain? I think
Tuj1 is restricted to immature neurons inside the brain ! though it stains
well with retinal ganglion cell....

I am also seeking for a nice marker for axon ...both normal and
regenerating...GAP-43 is one choice for regenerating axons...but a bit
focused on the growth cone, and the staining is not strong on transporting
proteins - due to the tiny amount? I am not sure how NF-200 or NF-96/160
work....

ANyone can suggest better markers for axon IHC staining? both normal and
regenerating.

____________________________________________________________________________
___________________
Axons stain nicely with beta 3 tubuline (TUJ). We've used the following
antibodies
Monoclonal anti-beta-tubulin isotype III from Sigma  1/1000  DAKO envision
(rivilation PAL or HRP) (ref : T8660)
or
Polyclonal  (rabbit)  antibody against neuronal class III beta-tubulin from
covance  research  commercialise  par  BABCO  Berkeley  antibody company au
1/1000 DAKO envision (rivilation PAL or HRP) (ref : PRB-435P)

We  used  paraffin  slices  of  PFA  fixed rat embryos and adult rat brain,
microwaving in citrate buffer. These antibodies also work nicely with IF

Good luck!
Nancy Walker
Molecular Biology Scientist

Sanofi-Synthelbo Research
B.P. 37 Labige Innopole
31676 LABEGE CEDEX FRANCE

nancy.walker <@t> sanofi-synthelabo.com
tel : (33)561004179  fax :(33)561004001



2009-03-05 



TF 

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

Message: 17
Date: Thu, 05 Mar 2009 16:17:47 +1100
From: Kim O'Sullivan <Kim.Osullivan <@t> med.monash.edu.au>
Subject: [Histonet] Natural Killer Cell antibody
To: histonet <@t> lists.utsouthwestern.edu
Message-ID: <130.194.114.97.1236230110 <@t> my.monash.edu.au>
Content-Type: text/plain; charset=UTF-8

Hi,

Does anyone out there know if there is a monoclonal anti human NK cell
antibody that is exclusive, ie does not also mark CD8 T cells etc?

Kim 



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

Message: 18
Date: Thu, 5 Mar 2009 08:15:35 +0200
From: Anne van Binsbergen <annigyg <@t> gmail.com>
Subject: Re: [Histonet] problem eyes
To: "Perry, Margaret" <Margaret.Perry <@t> sdstate.edu>
Cc: "histonet <@t> lists.utsouthwestern.edu"
	<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
	<f8332fbe0903042215s55668c0cq97bf1906abc0cc01 <@t> mail.gmail.com>
Content-Type: text/plain; charset=ISO-8859-1

do a search on the histonet archive
i posted a method quite a few years back
processed and cut eyes (human and piggy) on a daily basis for almost 10
years
go find it - it may help

patience, determination, thicker sections, 2 x pre-float out first on dist
water and then very dilute alcohol to manually tease out the crinkles, then
a gentle float on low ttemp water bath
if its too hot and the sections are too thin they will shatter on the warm
water
plenty of wax support in the block (use a bigger mould when you embed)

i am on vacation and saw this post by chance

unable to reply in too much detail

good luck

Annie (visiting in Africa)

2009/3/4 Perry, Margaret <Margaret.Perry <@t> sdstate.edu>

> Please help!  We have been trying to cut whole eyes from a pig.  They need
> to be nice enough for a publication.  We are having the devil of a time
> because no matter what we do there are wrinkles.  If we turn the waterbath
> up to stretch things out the retina detaches.  Do any of you have
> suggestions?  We uses Surgipath  EM400 for embedding paraffin and and
their
> infiltration medium.  Is this to soft? The lowest we can turn our
waterbath
> is 38 degrees C.
>
> Margaret Perry HT (ASCP)
> IHC Lab Manager Veterinary Science
> Animal Disease Research and Diagnostic Lab
> South Dakota State University
> Box 2175 North Campus Drive
> Brookings SD 57007
>
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>



-- 
Anne van Binsbergen (Hope)
Abu Dhabi
UAE


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

Message: 19
Date: Thu, 5 Mar 2009 02:19:19 -0600
From: Walzer Susan <Susan.Walzer <@t> HCAHealthcare.com>
Subject: [Histonet] RE: We are buying a new Tissue Processor and need
	imput
To: "Farnham, Lori" <farnhaml <@t> smha.org>,
	"histonet <@t> lists.utsouthwestern.edu"
	<histonet <@t> lists.utsouthwestern.edu>
Message-ID:
	
<4BF03F5404EBDE409AF9232DA74B9DED2AABBFA8CF <@t> FWDCWPMSGCMS09.hca.corpad.net>
	
Content-Type: text/plain; charset="us-ascii"

After 30 years in histology I say go with VIP for reliable processing.
(never had one fail or damage tissue)

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Farnham,
Lori
Sent: Wednesday, March 04, 2009 9:11 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] We are buying a new Tissue Processor and need imput

Hi All, 
We are a small Pathology Lab which processes a little over 7000 surgical
cases a year. We have a mixed variety of specimens (GI, GYN, skins,
etc).
We have been approved to buy a new tissue processor and are looking at
the Shandon Pathcentre and the Tissue Tek VIP 6. Any imput about these
processors would be greatly appreciated.
Thanks! 
Lori Ann Farnham, B.S.CT(ASCP)
Cytotechnologist
St. Mary's Hospital at Amsterdam
Phone 518-841-7296
e-mail farnhaml <@t> smha.org


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

Message: 20
Date: Thu, 5 Mar 2009 12:17:32 +0200
From: iskaliora <iskaliora <@t> bioacademy.gr>
Subject: [Histonet] staining brain vessels
To: histonet <@t> lists.utsouthwestern.edu
Cc: ????? ????????? <elenkons <@t> gmail.com>
Message-ID: <69CF6991-6EB5-4645-8A95-442EC8E9BCCC <@t> bioacademy.gr>
Content-Type: text/plain;	charset=UTF-8;	format=flowed;	delsp=yes

I was wondering if anybody might have an idea with the following  
problem we are experiencing:  we want to stain for blood vessels in  
sections of mouse brain.  Our experimental tissues have been fixed  
overnight in 4% paraformaldehyde and have been sitting in PBS since.   
We have tried staining with antibodies against desmin, SMA, and  
collagen but we get NO specific signal.  We recently tried a non-fixed  
mouse brain and got desmin to work immediately.  The problem is that  
we need to use the fixed brains because they are our experimental  
model and it would take too long (2 years to be exact) to generate the  
same samples.  If anybody has come across such a problem before, or  
has a specific protocol for vessels that works on PFA fixed brain, we  
would appreciate the suggestions!
thanks in advance!
Irini




-----------------------------------------------------
Irini Skaliora, PhD
Investigator Ca>= (Assistant Professor)
Developmental Biology Division
Biomedical Research Foundation of the Academy of Athens (BRFAA)
Soranou Efessiou 4
Athens 11527
tel. +30-210-6597203 (office)
tel. +30-210-6597482 (lab)
fax. +30-210-6597545
email: iskaliora <@t> bioacademy.gr







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

Message: 21
Date: Thu, 5 Mar 2009 08:00:49 -0500
From: "Pritchard, Michele" <pritchm <@t> ccf.org>
Subject: RE: [Histonet] Natural Killer Cell antibody
To: "Kim O'Sullivan" <Kim.Osullivan <@t> med.monash.edu.au>,
	histonet <@t> lists.utsouthwestern.edu
Message-ID:
	
<7CEB62F1535B9E44AD8A5FEFB2E10F6E021513C3 <@t> CCHSCLEXMB56.cc.ad.cchs.net>
Content-Type: text/plain; charset=us-ascii

CD56 (NCAM1) and CD16 (Fc gamma receptor III) are used to define human
NK cell populations.  Subsets of NK cells are distinguished from one
another based on 'bright' or 'dim' expression of these two markers.
Refer to Blood Rev. 20(3):123-37 (2006) for further information.

---mtp
Michele T. Pritchard, Ph.D.
Research Associate
Nagy Laboratory
Department of Pathobiology/NE40
Lerner Research Institute
Cleveland Clinic
9500 Euclid Avenue
Cleveland, OH 44195
 
phone:  216.444.8613
fax:  216.636.1493
 
email:  pritchm <@t> ccf.org
 
Lab location:
Lerner Research Institute
NE4-214
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Kim
O'Sullivan
Sent: Thursday, March 05, 2009 12:18 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Natural Killer Cell antibody

Hi,

Does anyone out there know if there is a monoclonal anti human NK cell
antibody that is exclusive, ie does not also mark CD8 T cells etc?

Kim 

_______________________________________________
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Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet


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

Message: 22
Date: Thu, 5 Mar 2009 07:13:15 -0600
From: "Bernice Frederick" <b-frederick <@t> northwestern.edu>
Subject: RE: [Histonet] problem eyes
To: <rjbuesa <@t> yahoo.com>, <histonet <@t> lists.utsouthwestern.edu>,
	"'MargaretPerry'" <Margaret.Perry <@t> sdstate.edu>,	"'Va Paula
Sicurello'"
	<vapatpxs <@t> yahoo.com>
Message-ID: <F172B048A7594C0297A50B79129CC8BD <@t> lurie.northwestern.edu>
Content-Type: text/plain;	charset="iso-8859-1"

We use it for fatty samples (we do lots of breast) and yes, it does work
well.
Bernice


Bernice Frederick HTL (ASCP)
Northwestern University
Pathology Core Facility
ECOGPCO-RL 
710 N Fairbanks Court
Olson 8-421
Chicago,IL 60611
312-503-3723


-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Rene J Buesa
Sent: Wednesday, March 04, 2009 3:28 PM
To: histonet <@t> lists.utsouthwestern.edu; MargaretPerry; Va Paula Sicurello
Subject: Re: [Histonet] problem eyes

Yes, really with any type of large (and small) sections. The liquid soap
will reduce the surface tension of water (more than heat) allowing the
sections to expand easily. Try it..
Reni J.

--- On Wed, 3/4/09, Va Paula Sicurello <vapatpxs <@t> yahoo.com> wrote:

From: Va Paula Sicurello <vapatpxs <@t> yahoo.com>
Subject: Re: [Histonet] problem eyes
To: "histonet <@t> lists.utsouthwestern.edu" <histonet <@t> lists.utsouthwestern.edu>,
"MargaretPerry" <Margaret.Perry <@t> sdstate.edu>, rjbuesa <@t> yahoo.com
Date: Wednesday, March 4, 2009, 4:23 PM

Really?  Liquid laundry soap?  Does this help with all wrinkles in large
specimens?


Paula Sicurello
VA Medical Center San Diego
Veterans Medical Research Foundation (VMRF) 
Core Microscope Facility, room B141
3350 La Jolla Village Dr., MC151
San Diego, CA 92161
858-552-8585 x2397


--- On Wed, 3/4/09, Rene J Buesa <rjbuesa <@t> yahoo.com> wrote:

> From: Rene J Buesa <rjbuesa <@t> yahoo.com>
> Subject: Re: [Histonet] problem eyes
> To: "histonet <@t> lists.utsouthwestern.edu"
<histonet <@t> lists.utsouthwestern.edu>, "Perry, Margaret"
<Margaret.Perry <@t> sdstate.edu>
> Date: Wednesday, March 4, 2009, 9:14 PM
> Add a few drops (4-5) of liquid
> detergent, but not dishwasher detergent (because it will
> dissolve the paraffin).
> reni J.
> 
> --- On Wed, 3/4/09, Perry, Margaret <Margaret.Perry <@t> sdstate.edu>
> wrote:
> 
> From: Perry, Margaret <Margaret.Perry <@t> sdstate.edu>
> Subject: [Histonet] problem eyes
> To: "histonet <@t> lists.utsouthwestern.edu"
> <histonet <@t> lists.utsouthwestern.edu>
> Date: Wednesday, March 4, 2009, 4:04 PM
> 
> Please help!  We have been trying to cut whole eyes
> from a pig.  They need to be
> nice enough for a publication.  We are having the
> devil of a time because no
> matter what we do there are wrinkles.  If we turn the
> waterbath up to stretch
> things out the retina detaches.  Do any of you have
> suggestions?  We uses
> Surgipath  EM400 for embedding paraffin and and their
> infiltration medium.  Is
> this to soft? The lowest we can turn our waterbath is 38
> degrees C.
> 
> Margaret Perry HT (ASCP)
> IHC Lab Manager Veterinary Science
> Animal Disease Research and Diagnostic Lab
> South Dakota State University
> Box 2175 North Campus Drive
> Brookings SD 57007
> 
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
> 
> 
> 
> 
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
> 







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





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

Message: 23
Date: Thu, 5 Mar 2009 21:31:28 +0800
From: "TF" <tifei <@t> foxmail.com>
Subject: Re: [Histonet] staining brain vessels
To: "iskaliora" <iskaliora <@t> bioacademy.gr>,	"histonet"
	<histonet <@t> lists.utsouthwestern.edu>
Cc: ????? ????????? <elenkons <@t> gmail.com>
Message-ID: <200903052131230910571 <@t> foxmail.com>
Content-Type: text/plain;	charset="utf-8"

hi, CD31 works great
in my section alpha-SMA also works

another way is to perfuse the brain with BSA-rhodamine.
you will get the fluorescence without the need of staining.


2009-03-05 



TF 



ed;6d::o< iskaliora 
ei