Cell kinetic analysis of intact rat colonic crypts by confocal microscopy and immunofluorescence
| Autor: | J. Jack Lee, Gary A. Glober, Angela Goodacre, Hideyuki Konishi, Michael Andreeff, Walter N. Hittelman, K. Fujita, Bernard Levin, Gideon Steinbach, Nicholas H. A. Terry |
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| Rok vydání: | 1996 |
| Předmět: |
Male
Pathology medicine.medical_specialty Mitotic index Colon Confocal Crypt Population Fluorescent Antibody Technique Cell Count Biology Immunofluorescence digestive system law.invention S Phase chemistry.chemical_compound Confocal microscopy law medicine Mitotic Index Animals Propidium iodide education education.field_of_study Microscopy Confocal Hepatology medicine.diagnostic_test digestive oral and skin physiology Cell Cycle Gastroenterology Histology Epithelial Cells digestive system diseases Rats Inbred F344 Rats chemistry |
| Zdroj: | Gastroenterology. 111(6) |
| ISSN: | 0016-5085 |
| Popis: | BACKGROUND & AIMS: Precise quantitative and spatial analysis of cell cycle-related biomarkers in colonic crypts is often vital for studies of colon carcinogenesis and cancer prevention. To overcome the limitations of histology, confocal laser microscopy of microdissected whole crypts was used to quantitate S phase and mitotic cells. METHODS: Microdissected distal colonic crypts were studied in a modified rat starvation refeeding model. S phase cells were labeled in vivo with 5- bromodeoxyuridine. Mitotic cells were labeled with MPM2 (antibody to mitosis-specific epitope) and also assessed for chromatin morphology with propidium iodide. Sequential optical crypt sections, produced by confocal microscopy, were digitally imaged. S phase labeling indices per whole crypt were also compared with those derived by conventional immunohistochemistry. RESULTS: S phase and mitotic cells were clearly discriminated without background staining. The labeled S phase cell number and fraction per whole crypt were significantly decreased with starvation and increased with refeeding. Variability in the labeling index between whole crypts analyzed by confocal microscopy was significantly smaller than between histological crypt sections. Consequently, the intervention contributed to 92.2% of the total variability of the labeling index in whole crypts but only to 59% of the variability in histological sections. CONCLUSIONS: Major limitations of histology are overcome by crypt microdissection and confocal microscopic analysis. The total crypt cell population as well as labeled M phase and S phase cells can be imaged, localized, and quantitated with improved precision. (Gastroenterology 1996 Dec;111(6):1493-500) |
| Databáze: | OpenAIRE |
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