This article is a US Government work and, as such, is in the public domain in the United States of America.
Version of Record online: 11 APR 2003
Published 2003 Wiley-Liss, Inc.
Cytometry Part A
Volume 53A, Issue 1, pages 9–21, May 2003
How to Cite
Price, O. T., Lau, C. and Zucker, R. M. (2003), Quantitative fluorescence of 5-FU–treated fetal rat limbs using confocal laser scanning microscopy and Lysotracker Red. Cytometry, 53A: 9–21. doi: 10.1002/cyto.a.10036
The research described in this article has been reviewed and approved for publication as an E.P.A. document. Approval does not necessarily signify that the contents reflect the views and policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
- Issue online: 11 APR 2003
- Version of Record online: 11 APR 2003
- Manuscript Accepted: 5 DEC 2002
- Manuscript Revised: 19 NOV 2002
- Manuscript Received: 19 APR 2002
- confocal microscopy;
- LysoTracker Red;
- fetal rat limbs;
- fluorescence quantification;
- marching cubes;
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).
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.
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.
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.