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Detecting Early Apoptosis in Whole Epithelial Sheets with a Caspase 3 Substrate, Phiphilux and Annexes V Using Confocal Microscopy

Published online by Cambridge University Press:  02 July 2020

Kathy K. H. Svoboda
Kathy K. H. Svoboda*
Affiliation:
Department of Biomedical Sciences, Texas A&M Health Science Center, Baylor College of Dentistry, Dallas, Tx, 75246
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Abstract

Many reagents have been developed recently to label living cells with substrates that will become fluorescent if an enzyme is active. The general principle is that the substrate will be taken up by living cells then detected only if the enzyme is active. These substrates work well with isolated individual cells, however, more difficulty can be encountered when studying whole tissues. Problems can range from substrate penetration into whole tissues to being able to detect the label effectively. We have used the chicken corneal epithelia for many studies, but the tissue is to thick to view with a conventional light microscope, therefore we have developed techniques using laser confocal microscopes to view this tissue in with a variety of techniques including in situ hybridization, immunohistochemistry, and vital dyes/stains.

Whole embryonic corneal epithelial sheets can be isolated without the basal lamina.

Type
Apoptosis in Health and Disease: Techniques for Detection and Biological Importance (Organized by M. Watanabe)
Information
Microscopy and Microanalysis , Volume 7 , Issue S2: Proceedings: Microscopy & Microanalysis 2001, Microscopy Society of America 59th Annual Meeting, Microbeam Analysis Society 35th Annual Meeting, Long Beach, California August 5-9, 2001 , August 2001 , pp. 600 - 601
Copyright
Copyright © Microscopy Society of America 2001

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References

1. Svoboda, KK (1992). “Embryonic corneal epithelial actin alters distribution in response to laminin.“ Invest. Ophthalmol. Vis. Sci. 33(2): 324–333.Google ScholarPubMed

2. Svoboda, KK (1991). “Intracellular localization of types I and II collagen mRNA and endoplasmic reticulum in embryonic corneal epithelia.” J Cell Sci 100(Pt 1): 23–33.Google ScholarPubMed

3. Svoboda, KKH, Orlow, DL, Ashrafeadeh, A and Jirawuthiworavong, G (1999). “Zyxin and vinculin distribution at the "cell-extracellular matrix attachment complex" (CMAX) in corneal epithelial tissue are actin dependent.ȝ Anat. Rec. ": 336–347.Google ScholarPubMed

4. Svoboda, KKH, Orlow, DL, Chu, CL and Reenstra, WR (1999). “ECM stimulated actin bundle formation in embryonic corneal epithelia is tyrosine phosphorylation dependent.” Anat. Rec. 254: 348–359.3.0.CO;2-5>CrossRefGoogle ScholarPubMed