Visualizing lens epithelial cell proliferation in whole lenses

Purpose To develop a means to image cells in S-phase of the cell cycle while preserving the anatomic relationships within the lens. Methods Mice were injected with the thymidine analog, EdU. Whole lenses were removed, fixed and permeabilized. Cells that had incorporated EdU into their DNA were chemically labeled using fluorescent azides and “click” chemistry. Double labeling was performed with antibodies to other antigens, like phospho-histoneH3, a marker of mitotic cells. The position of labeled cells and lens anatomy was viewed using a simple device to position and flatten the lens. Results The nuclei of cells in S-phase of the cell cycle were intensely stained without the use of antibodies. Stained cells were readily localized with reference anatomic landmarks, like the transition zone. Whole lenses could be assayed by rotating the lens on the microscope stage. Double-labeling permitted the co-localization of markers in cycling cells. Conclusions EdU labeling of whole lenses provides a simple, rapid and sensitive means to analyze lens epithelial cell proliferation in the anatomic context of the whole lens.