Fluorescence microscopy has long been used for qualitative characterization of various parameters such as subcellular distribution of proteins, lipids, nucleic acids, and ions. However, quantification of these parameters is complicated by a variety of optical, biological, and physical factors. In the last decade, the progress achieved with powerful softwares and digital image processing systems has facilitated the development of fluorescence immunohistochemistry (FIHC) into a widely used quantitative assay (quantitative-FIHC or Q-FIHC). We describe here a rapid and sensitive Q-FIHC assay based on the use of a laser scanning confocal microscope and advanced image analysis softwares (Zeiss semi automatic LSM 510 and fully automatic Axiovision 4.4) for the detection and quantification of fluorescent intensity in human corneal tissues and cells obtained from small clinical samples. We have used this methodology to characterize and quantify the gene expression profile of p63 and its DeltaNalpha isoform, specific markers of human limbal stem cells. The validity of this method was evaluated through comparative studies with conventional approaches suggesting no significant differences and providing an alternative technique to traditional methods. Since Q-FIHC requires at least 20-fold less cells than traditional techniques, we have adopted it as the main quality control for our limbal cultures destined to clinical application.
Copyright (c) 2006 Wiley-Liss, Inc.