A Simple Method to Dissect, Orientate, and Visualize the Murine Limbal Stem Cell Niche with Cornea and Conjunctiva Attached.

Abstract

The limbus is a narrow tissue intersection between the cornea and conjunctiva which is purported to harbor stem cells (SCs) that replenish the corneal epithelium throughout life. Damage to these cells can result in debilitating visual consequences. To date, various immunohistochemical methods have been employed to investigate limbal morphology and identify SC location to improve their isolation for therapeutic use. However, none of these methods preserve tissue integrity and orientation, nor do they incorporate adjacent conjunctiva as a contiguous ocular surface for analyses. In this chapter, we provide a methodology to overcome these limitations by integrating a unique dissection technique along with a tissue clearing strategy to enable the detection of morphological features within the limbal SC niche in different locations across its circumference. The morphological and biochemical details acquired from such investigations will heighten the current understanding of changes in tissue architecture in healthy and diseased corneas and in those that have been treated with biologicals, pharmacological, and/or surgical interventions.