Visualization of interstitial cells of Cajal in living, intact tissues.
Interstitial cells of Cajal (ICC) appear to be a major element in pacing and signal transmission in the gastrointestinal tract. A prominent problem in the study of ICC has been the difficulty in observing them in intact tissues. We used several methods to visualize living ICC in freshly-dissected tissues: (1) Placing small crystals of the lipophilic dye DiI in the submucosal-circular muscle border in the mouse colon resulted in the labeling of living ICC-like cells. Two main morphological cell types, bipolar and multipolar, were noted. The DiI stain could be converted into a stable, electron-opaque product. Electron-microscopic observations showed that the labeled cells had the typical appearance of ICC reported in previous studies. (2) Living ICC in the region of the myenteric plexus (ICC-MP) in the small intestines of mice and guinea-pigs were observed with Nomarski optics. This enabled the visualization of ICC in living tissues, and the impalement of the cells with Lucifer yellow-filled microelectrodes. The dye-labeled cells had the morphological features of ICC-MP, and about 30% of them were found to be dye coupled to 1-21 other ICC. The identity of the cells as ICC was verified by electron-microscopy following photoconversion, and by c-kit immunohistochemistry. (3) Living ICC were labeled with a c-kit antibody that does not require tissue fixation. This resulted in the fluorescent staining of the entire ICC network. Single cells were labeled by dye injection, which provided a detailed picture of ICC morphology. This method was found to be suitable for a wide range of tissues. We expect that these three methods for identifying ICC in intact, living tissues will be useful for physiological and pharmacological investigations of ICC in a variety of gastrointestinal tissues.
Hanani
M
M
Hebrew University-Hadassah School of Medicine, Jerusalem, 91240, Israel. hananim@cc.huji.ac.il
Belzer
V
V
Rich
A
A
Faussone-Pellegrini
S M
SM
D000818:Animals; D000911:Antibodies, Monoclonal; D002232:Carbocyanines; D004064:Digestive System / Q000166:cytology; D005260:Female; D006168:Guinea Pigs; D006651:Histocytochemistry / Q000295:instrumentation; D007089:Image Enhancement / Q000379:methods; D007150:Immunohistochemistry; D007422:Intestines / Q000737:chemistry / Q000166:cytology* / Q000648:ultrastructure; D007546:Isoquinolines; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008854:Microscopy, Electron; D055095:Optics and Photonics; D019009:Proto-Oncogene Proteins c-kit / Q000032:analysis / Q000276:immunology; D013194:Staining and Labeling / Q000379:methods*
D000911:Antibodies, Monoclonal; C101089:CM-DiI; D002232:Carbocyanines; D007546:Isoquinolines; C017475:lucifer yellow; D019009:Proto-Oncogene Proteins c-kit