[Histonet] Drying Slides before Filing

From:"Kopczynski, Charlotte"

Just wanted to thank those who responded to my question about drying slides
before filing.  Was wondering if there are more out there who can share what
they are doing.  Method of drying and length of time....
Charlotte Kopczynski,HTL(ASCP)
Baycare Pathology Manager
Phone: 727-461-8246
Fax:   727-462-7597

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From: histonet-request@lists.utsouthwestern.edu
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To: histonet@lists.utsouthwestern.edu
Subject: Histonet Digest, Vol 9, Issue 40

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Today's Topics:

   1. Kerry L Crabb/PharmRD/GSK is out of the office.
   2. Using Cy2 immunofluorescence? (pcfanti@buffalo.edu)
   3. Re: Bacteria in eye lens (Linda Jenkins)
   4. RE: FW: Salt-split skit technique for immunofluorescence
      (Barry R Rittman)
   5. Re: alcian blue pH (Geoff McAuliffe)
   6. Atherosclerosis and freshly cut tissue (Douglas A Gregg)
   7. Open Position - IP Supervisor (aviancourt@propathlab.com)
   8. EDTA separation MO (Barry R Rittman)


Message: 1
Date: Thu, 26 Aug 2004 10:45:28 -0400
From: kerry.l.crabb@gsk.com
Subject: [Histonet] Kerry L Crabb/PharmRD/GSK is out of the office.
To: histonet@lists.utsouthwestern.edu
Content-Type: text/plain; charset=us-ascii

I will be out of the office starting  26-Aug-2004 and will not return until

Contact Eve about necropsy issue and contact Teresa about histology issues.
I will respond to your message when I return.


Message: 2
Date: Thu, 26 Aug 2004 11:02:23 -0400
From: pcfanti@buffalo.edu
Subject: [Histonet] Using Cy2 immunofluorescence?
To: histonet@lists.utsouthwestern.edu
Message-ID: <1093532543.412dfb7f6c865@mail3.buffalo.edu>
Content-Type: text/plain

We are using a cy2 immunofluorescent secondary antibody for IHC, and 
we would like to know if there are any complications using it on a 
biotin conjugated primary. We are not sure if there will be 
interferance with the signal or if there will be no sites on the 
primary ab for the secondary to bind. I appreciate your help.
Peter Fanti
University at Buffalo 
OB/GYN department


Message: 3
Date: Thu, 26 Aug 2004 11:14:56 -0400
From: Linda Jenkins 
Subject: [Histonet] Re: Bacteria in eye lens
To: histonet@lists.utsouthwestern.edu
Content-Type: text/plain; charset="us-ascii"; format=flowed

Dear Angel,
         Is this an artificial lens implant that is inserted during 
cataract surgery?  There have been numerous problems with bacterial growth 
with these implants.  It was theorized that the design of the lens implant 
was partly responsible for the bacterial growth and when a textured surface 
was added to the design, the bacteria was eliminated.  Again, it was 
theorized that the textured surface promoted quicker cellular attachment.
         Hope you find this useful,

Linda Jenkins, HT
Clemson University
Dept. of Bioengineering
Clemson, SC 29634-0905


Message: 4
Date: Thu, 26 Aug 2004 10:13:58 -0500
From: "Barry R Rittman" 
Subject: RE: [Histonet] FW: Salt-split skit technique for
To: "histonet" 
Content-Type: text/plain;	charset="us-ascii"

Are you referring to the separation of epithelium from the underlying
connective tissue by splitting at the basement membrane?
If so I can send you the EDTA separation technique.

-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu
[mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of Sharon
Sent: Thursday, August 26, 2004 8:35 AM
To: Histonet (E-mail)
Subject: [Histonet] FW: Salt-split skit technique for immunofluorescence

> -----Original Message-----
> From:	Sharon Allen 
> Sent:	August 23, 2004 9:37 AM
> To:	Histonet (E-mail)
> Subject:	Salt-split skit technique for immunofluorescence
> Hi,
> Does anyone have a method for the "Salt-split skin technique for
> immunofluorescence"?
> I would appreciate any help with this method.
> Thanks
> Sharon Allen
> sallen@hsc.mb.ca
> Winnipeg, Manitoba, Canada

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Message: 5
Date: Thu, 26 Aug 2004 11:18:27 -0700
From: Geoff McAuliffe 
Subject: Re: [Histonet] alcian blue pH
To: Tom Schaer 
Cc: Histonet@lists.utsouthwestern.edu
Message-ID: <412E2973.9080706@umdnj.edu>
Content-Type: text/plain; format=flowed; charset=ISO-8859-1

Hi Tom:

    Alcian Blue will stain the glycosaminoglycans in the extracellular 
matrix, it won't show much cellular morphology. Still, the traditional 
method is 1% Alcian Blue in 3% acetic acid for a pH of 2.5. At 2.5 both 
sulfated and carboxylated GAGs are stained. A red nuclear stain, Scarba 
red or nuclear fast red, gives a nice contrasty counterstain.
    You might try 0.05% Toluidine blue in dist water with a pH of about 
4.0 for 10-15 minutes, dist water rinse and dehydration in 3 changes of 
actone followed by clearing in fresh xylene. Nice metachromasia of 
extracellular matrix and you will see some cellular morphology as well.


Tom Schaer wrote:

>Greetings List:
>I would like to stain some intervertebral disc x-sections (rat,
decalcified) with alcian blue.  Interest in cellular morphology of nucleus
pulposus, transition and annulus cells.
>What pH of the stain should I use.
>Many thanks, 
>Thomas P. Schär, VMD
>Asst  Prof  School of Biomedical Engineering, Drexel University
>& Comparative Orthopaedic Research & Tissue Engineering
>Department of Clinical Studies
>University of Pennsylvania New Bolton Center
>382 West Street Road
>Kennett Square, PA 19348
>tel. 610-444-5800 (x6261 office)
>tel. 610-444-5800 (x6131 lab)
>fax. 610-925-8100
>Histonet mailing list

Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029 


Message: 6
Date: Thu, 26 Aug 2004 11:34:00 -0400
From: Douglas A Gregg 
Subject: [Histonet] Atherosclerosis and freshly cut tissue
To: histonet@lists.utsouthwestern.edu
Cc: syedab@totalise.co.uk
Message-ID: <20040826.113401.3676.0.d.gregg@juno.com>
Content-Type: text/plain; charset=us-ascii

I would suggest that in future studies to try PLP fixative
(paraformaldehyde-lysine-periodate). I have had good experience with a
number of antigens that I have been able to preserve in wet tissue for
years. I work mainly with viruses. About half of the viral antigens tried
could be preserved for years. The other could only stand a few days to a
couple of weeks in fixative before losing their antigenicity. Retrieval
is needed in almost every case but staining can be as good as fresh
tissue even years later. Formalin fixed tissue usually failed completely
after a year even with the most robust antigens. PLP will turn yellow and
stain the tissues yellow to some extent after a few weeks or months, but
don't worry about it. This does not seem to affect the immunostaining.  I
might add that with some antigens it works better to leave the tissue in
the wet PLP until needed and then embed what you need to cut shortly
before immunostaining. I have lost staining in paraffin blocks in as
little as one month when the wet tissue re-embedded was fine. My record
is 12 years. 

Best of Luck,
Douglas Gregg
Veterinary Pathologist
Plum Island Animal Disease Center
Greenport,k NY 11944


Message: 7
Date: Thu, 26 Aug 2004 10:41:43 -0500
From: aviancourt@propathlab.com
Subject: [Histonet] Open Position - IP Supervisor
To: Histonet@lists.utsouthwestern.edu
Content-Type: text/plain;	charset="iso-8859-1"

ProPath, a high volume Anatomic Pathology practice in Dallas, Texas has an
opening for a Supervisor in the Immunohistochemistry Laboratory.  The
position reports directly to Dr. Rodney Miller.  Candidates with HT/HTL and
QIHC registry and supervisory experience are encouraged to send a resume to:

8267 Elmbrook Dr, Suite 100
Dallas, TX 75247
Fax: 214/237-1820
Email: jobs@propath.com 


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Message: 8
Date: Thu, 26 Aug 2004 11:31:26 -0500
From: "Barry R Rittman" 
Subject: [Histonet] EDTA separation MO
To: "histonet" 
Content-Type: text/plain;	charset="us-ascii"

General procedure

The use of EDTA chelates calcium in the basement membrane allowing the
basement membrane to split and thus separates the epidermis from the


1.	Carefully shave hair from the skin using a stiff backed
commercial blade. The blade should first be rinsed in acetone to remove
any grease that may be present.

It is best to use commercial blades and to try not to cut the epidermis
but to just remove the hair.  If super sharp blades are used then the
epidermis generally will become cut in areas and this affects subsequent
treatment. If skin is from animal ears then it is best to first separate
both sides at the cartilage plate.

Remove excess fat and connective tissue as this will decrease
penetration of EDTA.


2.	Dip for few seconds in 70% ethanol followed by 2- 3 changes of
phosphate buffered saline (PBS). This to remove debris or loose hair
that may be on the skin and does not have any fixing action. 


3.	Place in a small closed jar containing 3mM EDTA in PBS (see
below) in a shaking water bath for 2 hrs at 37 degrees C. For a piece of
abdominal skin 1 by 1 cm probably use around 40 - 50 ml. of solution. 

In addition, the jar should be removed every half hour and briefly
shaken to ensure all pieces of skin remain separate from each other.


4.	Pour contents of jar into a shallow Petri dish with a small
amount of fluid.


5.	Arrange pieces so that epithelium side is uppermost.


6.	Using a dissecting microscope at low power and Dumont style #7
forceps, hold the edge of the connective tissue onto the Petri dish with
one pair of forceps. With the second pair gentle ease an edge of
epithelium. Once an edge is free then the epithelium can be peeled
taking care to go in one direction. This takes a little practice. Use
pieces of skin from an understanding relative to start with until you
become proficient.


7.	The separated epithelium should float on the solution. It can be
lifted using a glass hockey stick and treated as a free floating
section, with the remnants of the basement membrane down.  If there are
tears in the epithelium it will tend to allow fluid to gain access to
the surface and affect staining in that area.


8.	The epidermal sheet can be floated onto PBS and used for cell
separation, histochemistry, immunohistochemistry etc. It is important
for any staining that sheets are not submerged in reagents but that they
float on the reagents with the surface of epidermis uppermost. This
provide more uniform staining.


 3 mM EDTA

3 mM disodium EDTA (0.558 gms disodium EDTA in phosphate buffered saline
with pH adjusted to 7.3 to 7.4. 

Separation of some mucosae such as dog palate and thick skin samples may
require 10-20 mM EDTA instead of 3mM or longer times. These conditions
do however decrease the uniformity of staining.




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