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Quantitative fluorescence of 5‐FU–treated fetal rat limbs using confocal laser scanning microscopy and Lysotracker Red
Author(s) -
Price Owen T.,
Lau Christopher,
Zucker Robert M.
Publication year - 2003
Publication title -
cytometry part a
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.316
H-Index - 90
eISSN - 1552-4930
pISSN - 1552-4922
DOI - 10.1002/cyto.a.10036
Subject(s) - confocal microscopy , confocal , fluorescence , fluorescence microscope , confocal laser scanning microscopy , laser microscopy , microscopy , biophysics , fetus , chemistry , materials science , biology , pathology , medicine , optics , microbiology and biotechnology , physics , pregnancy , genetics
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 in tissues resulting from phagocytosis of apoptotic bodies by neighboring cells. LT was shown previously to be an indicator of cell death in a manner similar to other standard assays (Annexin, terminal dUTP nick end labeling, Nile blue sulfate, neutral red, and acridine orange). Methods LT fluorescence in fetal rat hindlimbs at gestational day 14 was measured 8 h after administration of the teratogen, 5‐fluorouracil (5‐FU), with the use of confocal laser scanning microscopy (CLSM). Four dose levels of 5‐FU (0, 20, 30, and 40 mg/kg) were studied. The preparation technique involved staining with LT, paraformaldehyde fixation, methanol dehydration, and clearance with benzyl alcohol and benzyl benzoate. After 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) hindlimbs. After 5‐FU treatment, highly fluorescent regions appeared in the progress zone (PZ) of the limb. A dose‐dependent response to 5‐FU treatment was observed. Compared with controls, hindlimbs treated with 20, 30, and 40 mg/kg of 5‐FU exhibited more fluorescence within the highly proliferative PZ. These results showed a dose–response relation between 5‐FU exposure and LT uptake. Conclusions We found that three‐dimensional volumetric regions indicating a high level of fluorescence in the embryonic limb bud can be quantified with three different computer analysis programs. The combination of a sample preparation procedure that clears tissue, a CLSM technique that addresses the equipment variables, and an application of statistical population analysis procedures enabled the visualization and quantification of fluorescence in entire fetal rat hindlimbs that were approximately 500 μm in thickness. Cytometry Part A 53A:9–21, 2003. Published 2003 Wiley‐Liss, Inc.