[Histonet] EDTA separation MO

From:"Barry R Rittman"

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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