The search for ganglion cells in frozen section on open biopsies is aided by
a non-specific esterase (hexazotizied pararosanilin) method when the biopsy
is post-fixed in acetic alcohol before staining. Ganglion cells are strongly
positive for this enzyme method.

Section:
Prefixed cryostat section.

Solution:
1. Substrate solution -
alpha naphthyl acetate    50 g
acetone                    5 ml

2. Buffer solution -
Disodium hydrogen phosphate   2.83 g
DH2O                           100 ml

3. Sodium nitrite solution -
Sodium nitrate                400 ml
DH2O                           10 ml

4. Pararosanilin-HCl stock solution -
Pararosanilin HCl             2 g
2 M HCl                      50 ml
Heat gently, cool to room temperature and filter.

5. DH2O

Solution Preparation:
Solution 1          0.25 ml
Solution 2          7.25 ml
Solution 3           0.4 ml
Solution 4           0.4 ml
Solution 5           2.5 ml

Method:
1. After suitable fixation, bring sections to water.
2. Incubate in solution at 37C for 2-20 min.
3. Wash in running water.
4. Counterstain in 2% methyl green (chloroform extracted).
5. Wash well in running water.
6. Dehydrate rapidly through fresh alcohol to xylene and mount in
sythentic resin.

Results:
Esterase-reddish brown
Nuclei-green

Reference:
Davies & Ornstein, 1959.


Eric C. Kellar
University of Pittsburgh Medical Center







-----Original Message-----
From: Sebree Linda A.
To: 'Histonet'; 'IPOX'
Sent: 9/1/99 3:04 PM
Subject: ganglia identification in colonoscopy biopsies

Hi Histonetters and IPOXers,

One of our pathologists has asked us to find a way to identify the
presence
of ganglia in frozen sections of colon biopsies.  Colonoscopy is
performed
to identify Hirschsprung's disease.  The idea is to sample sections of
colon
and perform quick stains to access the presence or absence of ganglia.
I
don't know of any immunohistochemistry procedure that can be done within
the
30-45 minutes time frame.  All of the biopsies would be assessed while
the
patient is on the O.R. table.

So....does anyone out there know of a histochemical, enzyme or
immunohistochemical procedure that could be used?

Your help, as always, is greatly appreciated,

Linda A. Sebree, HT
University of Wisconsin Hospital & Clinics
Immunohistochemistry/In Situ Hybridization Laboratory
D4/218-2472
600 Highland Avenue
Madison, WI  53792-2472


(608)265-6596
FAX: (608)263-1568