Donna,
I asked histonet to respond directly to you on this but they did not, so I
am forwarding this to you.  To sign up for the histonet list server, send an
email message to histonet@pathology.swmed.edu and in the subject line write
subscribe to get off send an email message to the same place and in the
subject line write unsubscribe
Patsy Ruegg
-----Original Message-----
From: Barry Rittman
To: histonet@Pathology.swmed.edu
Sent: 6/4/99 4:07 PM
Subject: Re: epithelial sheets

Donna,
            I assume that you are using backskin and that you are
removing the
fur before placing in the EDTA solution. How are you removing the fur?
How long
in EDTA? What size areas of skin are you staining?
We remove fur using wax and then rinsing the skin briefly in 70% ethanol
followed by 2-3 rinses of sterile saline. Any fat desposits etc removed.
EDTA was 3mM in PBVS pH 7.35. for 2 hours at 37 on shaking water bath
and then
separated using a dissecting microscope. Sheets into PBS and then
stained etc.
Sheets were floated connective tissue side down on the solutions. If any
solution creeps over ffrom the underside to the top surface due to tears
etc.
in the skin  then staining will be patchy  in those areas.

For keeping sections flat in glycerol there is a problem. When in
glycerol,
bubbles form after 24-48 hours and are a pain to remove. You can mount
in
glycerol gelatine and this works quite well. However, need to use a
hotplate
while mounting, remove as much fluid as possible and add the
glycerin-gelatine.
As soon  as the coverglass is placed on the slide, need to place a
weight on
the surface to keep it flat while it is setting. Sets in about 3-5
minutes.
While a weight on the surface onf the coverslip will help when mounting
in
glycerol, it's difficult to get the sheets flat over the entire surface.
If you
are counting numbers of  Langerhans cells and using a large enough sheet
of
epidermis, this may not be a problem as the distribution of L cells in
epidermis is relatively uniform in mice.
Please call me if you need more details, I have stained lots of these
sheets
for Langerhans cells for Ia, ATPase, b -glucuronidase, esterase etc. so
much so
that the novelty has worn off.
I will be happy to discuss this at length if you wish, call me .
Barry

Patsy.Ruegg@UCHSC.edu wrote:

> Kind friends,
>
> I am posting this for someone else.  If you can help this person,
please
> respond to them directly as they apparently are not on histonet.  Tell
them
> how to subscribe to histonet while you are at it please.
>
> I would like some help with staining problems.  I am removing
epidermal
> sheets from mouse skin using EDTA and then staining for Langerhans
cells
> (biotinylated anti-Ia primary antibody with ABC/DAB detection). I'm
having
> two kinds of problems:
> 1.      Cells in the center of the sheet don't stain.
> 2.      The epithelial sheets don't lie flat when I mount them on
slides in
> glycerol.
>
> Can anyone give me some advice on how to correct these problems?  I
would
> really appreciate some help.
> Donna Kusewitt
>
> Donna F. Kusewitt, DVM, PhD
> Research Associate Professor
> Department of Cell Biology and Physiology
> Room 149 BMSB
> 915 Camino de Salud, NE
> School of Medicine
> University of New Mexico
> Albuquerque, NM  87131
> Telephone: 505-272-1302
> FAX: 505-272-9105
> Email: dkusewitt@salud.unm.edu <mailto:dkusewitt@salud.unm.edu>