PubMed · 6372610
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E J Holborow, G D Johnson. 1983. Standardization in immunofluorescence.. https://doi.org/10.1111/j.1749-6632.1983.tb22188.x
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CONTEXT: -c-Kit-positive interstitial cells of Cajal (ICC) appear to play a key role in the normal motility function and development of intestine. Nitric oxide is considered to be the most important messenger of inhibitory nonadrenergic, noncholinergic nerves in the enteric nervous system. OBJECTIVES: The aims of this study were to examine the distribution of nitrergic innervation and ICCs in normal human bowel and to demonstrate interconnections between ICCs and nitrergic nerves and smooth muscle fibers using histochemical and immunohistochemical double-staining methods with a whole-mount preparation technique and confocal laser scanning microscopy. METHODS: Full-thickness small and large bowel specimens were obtained at autopsy from 18 children who died of nongastrointestinal diseases. A whole-mount preparation was performed for all specimens, and double staining was carried out with nicotinamide adenine dinucleotide phosphate (reduced form, NADPH)-diaphorase and c-Kit immunohistochemistry. Double immunofluorohistochemistry with neuronal nitric oxide synthase and c-Kit using confocal laser scanning microscopy was also performed in all specimens. RESULTS: The whole-mount preparation facilitated 3-dimensional visualization of the meshlike network of NADPH-diaphorase-positive nerve fibers in the myenteric plexus surrounded by a reticular network of c-Kit-positive ICCs. The dense c-Kit-positive cellular network located between longitudinal and circular muscle layers and at the innermost part of circular muscle layer intermingled with the myenteric plexus. Short, fine processes of ICCs made connections with the muscle fibers and c-Kit-positive cells. CONCLUSIONS: The development of double-NADPH-diaphorase histochemistry and c-Kit immunohistochemistry staining technique in a whole-mount preparation provides an easy and useful method for investigating the association between c-Kit-positive cellular network and nitrergic neuronal network in the human bowel wall. The characteristic profiles of the c-Kit-positive cellular network and nitrergic neuronal network and their relationship with the smooth muscle fibers provide a morphologic basis for investigating intestinal motility disorders.