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QUANTITATIVE FLUORESCENCE OF 5-FU-TREATED FETAL RAT LIMBS USING CONFOCAL LASER SCANNING MICROSCOPY AND LYSOTRACKER RED
Citation:
Price, O. T., C S. Lau, AND R M. Zucker. QUANTITATIVE FLUORESCENCE OF 5-FU-TREATED FETAL RAT LIMBS USING CONFOCAL LASER SCANNING MICROSCOPY AND LYSOTRACKER RED. CYTOMETRY. Alan R Liss, Inc, New York, NY, Part A 53A(1):9-21, (2003).
Description:
Background: LysoTracker Red (LT) is a paraformaldehyde fixable probe that concentrates into acidic compartments of cells and tissues. After cell death a high level of lysosomal activity (acidic enzyme) is expressed resulting from phagocytosis of apoptotic bodies by neighboring cells. Previously, LT has been shown to be an indicator of apoptotic cell death in a manner similar to other standard assays.Methods: Apoptosis in fetal rat hind limbs at gestational day (GD) 14 was measured 8 hours after an administration of the teratogen, 5-fluorouracil (5-FU) using confocal laser scanning microscopy (CLSM). Four dose levels of 5-FU (0, 20, 30, 40 mg/kg) were studied. The preparation technique involved staining with LT, paraformaldehyde fixation, methanol dehydration, and benzyl alcohol/benzyl benzoate clearance. Following this treatment, the limb was nearly transparent and ready for CLSM analysis. Results: LT staining was observed in specific regions undergoing apoptosis in normal (control) hind limbs. After 5-FU treatment, highly stained regions appeared. A dose-dependent response to 5-FU treatment was observed. Compared to controls, hind limbs treated with 20, 30 and 40 mg/kg of 5-FU exhibited, respectively, at least 10, 40, and 80 times more fluorescence within the highly proliferative progress zone. These results show a dose-response relationship between 5-FU exposure and LT uptake. Conclusions: It has been shown that three-dimensional (3D) volumetric regions indicating a high level of fluorescence in the embryonic limb-bud can be quantified. The combination of a unique sample preparation procedure, confocal laser scanning microscope technique, and a unique application of statistical population analysis procedures enabled the visualization and quantification of apoptosis in fetal rat hind limbs (approximately 500 m in thickness).