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Journal of Histochemistry and Cytochemistry, Vol. 46, 1223-1232, November 1998, Copyright © 1998, The Histochemical Society, Inc.

ARTICLE

Choline Acetyltransferase (ChAT) Immunoreactivity in Paraffin Sections of Normal and Diseased Intestines

Elyanne M. Ratcliffea, Derek J. deSab, Michael F. Dixonc, and Ronald H. Steada,d
a Department of Pathology, McMaster University, Hamilton, Ontario, Canada
b Department of Pathology and Laboratory Medicine, B.C. Children's Hospital, Vancouver, British Columbia, Canada
c Academic Unit of Pathology, University of Leeds, Leeds, United Kingdom
d Holburn Biomedical Corporation, Bowmanville, Ontario, Canada

Correspondence to: Ronald H. Stead, Holburn Biomedical Corp., 30 Caristrap Street, Bowmanville, ON, Canada L1C 3Y7..

There is increasing interest in localizing nerves in the intestine, especially specific populations of nerves. At present, the usual histochemical marker for cholinergic nerves in tissue sections is acetylcholinesterase activity. However, such techniques are applicable only to frozen sections and have uncertain specificity. Choline acetyltransferase (ChAT) is also present in cholinergic nerves, and we therefore aimed to establish a paraffin section immunocytochemical technique using an anti-ChAT antibody. Monoclonal anti-choline acetyltransferase (1.B3.9B3) and a biotin–streptavidin detection system were used to study the distribution of ChAT immunoreactivity (ChAT IR) in paraffin-embedded normal and diseased gastrointestinal tracts from both rats and humans. Optimal staining was seen after 6–24 hr of fixation in neutral buffered formalin and overnight incubation in 1 µg/ml of 1.B3.9B3, with a similar distribution to that seen in frozen sections. In the rat diaphragm (used as a positive control), axons and motor endplates were ChAT IR. Proportions of ganglion cells and nerve fibers in the intramural plexi of both human and rat gastrointestinal tracts were also ChAT IR, as well as extrinsic nerve bundles in aganglionic segments of Hirschsprung's disease. Mucosal cholinergic nerves, however, were not visualized. In addition, non-neuronal cells such as endothelium, epithelium, and inflammatory cells were ChAT IR. We were able to localize ChAT to nerves in formalin-fixed, paraffin-embedded sections. The presence of ChAT IR in non-neuronal cells indicates that this method should be used in conjunction with other antibodies. Nevertheless, it proves to be a useful technique for studying cholinergic neuronal distinction in normal tissues and pathological disorders. (J Histochem Cytochem 46:1223–1231, 1998)

Key Words: choline acetyltransferase, immunocytochemistry, cholinergic nerves, enteric nervous system, intestine, Hirschsprung's disease


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