Visualization of Conjunctival Goblet Cell Actin Cytoskeleton and Mucin Content in Tissue Whole Mounts

Abstract

The purpose of the work was to visualize goblet cells, their cytoskeletons, and their content in tissue whole mounts of conjunctiva using confocal microscopy. Conjunctival tissue from rat, mouse, rabbit, and human were excised, fixed in 4% paraformaldehyde, and incubated either with rhodamine-labeled phalloidin to label filamentous actin or in combination with fluoresceinated lectins to label carbohydrates on goblet cell mucins. Tissues were evaluated by confocal microscopy. Phalloidin labeling of excised tissue facilitated visualization of goblet cells within the stratified epithelium of the conjunctiva. Phalloidin labeled the perimeter of the goblet cells, so that either in optical section or three-dimensional constructs of optical sections, the goblet cells appeared as plump empty clusters of cells in rat and mouse and as individual plump empty cells in rabbit and human tissues. Double labeling of excised tissue with lectins demonstrated that the plump cells visualized by labeling with phalloidin bound the label and were indeed goblet cells. At very high resolution one could see individual mucin packets within the goblet cells. Variation in intensity of lectin labeling of these mucin packets both between cells and within cells was evident. Streams of labeled mucin emanating from apical surfaces of goblet cells could be visualized using the fluoresceinated lectins. The labeling of excised conjunctiva with fluorochrome-conjugated phalloidin allows visualization by confocal microscopy of goblet cells within the stratified epithelium. Double labeling with fluorochrome-conjugated lectins allows visualization of their content. These methods may be useful in studies of goblet cell differentiation, mucin content and secretion.