Whole mount staining of lenses for visualization of lens epithelial cell proteins

Abstract

Whole mount imaging of the lens allows for high spatial resolution visualization of lens epithelial structures by using small molecule fluorescent probes. However, the visualization of specific proteins in lens epithelial cells within whole lenses remains a challenge as the capsule that surrounds the lens does not allow penetration of antibodies. Here we describe a whole mount imaging method that allows us to overcome this challenge by digesting the lens capsules of paraformaldehyde fixed lenses using collagenase. This method enables the penetration of antibodies for effective visualization of proteins in the epithelium of whole lenses. A limitation to lens whole mount imaging is the ability to visualize specific proteins as the collagen capsule surrounding the lens impedes the penetration of antibodies This protocol helps overcome this limitation by a light collagenase digestion of the capsule of fixed lenses prior to immunostaining This method allows for the imaging of specific proteins in the epithelium of the whole lens tissue

Methodologies to unlock the molecular expression and cellular structure of ocular lens epithelial cells

“…However, large antibodies do not readily penetrate the lens capsule. We have previously detailed a method to immunostain the whole lens after incubation in collagenase to partially digest the lens capsule and facilitate antibody penetration ( Patel et al, 2021 ). Whole mount staining maintains the 3D structure of tissue, but imaging whole mount lenses requires repositioning of the lens to visualize the various regions of the epithelium.…”

“…Whole mount staining maintains the 3D structure of tissue, but imaging whole mount lenses requires repositioning of the lens to visualize the various regions of the epithelium. Excessive disruption of the lens capsule for antibody labeling may alter the basal surface of lens epithelial cells and lead to cellular disorganization ( Patel et al, 2021 ).…”