An in situ method for the examination of calcium-dependent proteolysis

Proteases are involved in a multitude of cellular processes that are critical for the maintenance of normal cell function, and their aberrant activity has been linked to a large number of diseases. Calcium-dependent proteases (calpains) are found in cells distributed throughout the brain, and their activity contributes to normal and abnormal brain function. A limitation with common approaches to studying the activity of calpain is the requirement for homogenization of tissue samples, which limits the ability to resolve the spatial location of protease activity, and which also introduces the possibility of interaction with endogenous inhibitors that would have otherwise been kept spatially separated in vivo. We present a simple method for the investigation of protease activity that provides better spatial resolution than alternatives, and that alleviates the concern of protein interactions in homogenate. We examined calcium-dependent proteolysis in tissue sections by observation of a fluorescence signal produced by fragmentation of a casein substrate embedded in an agarose gel solution that covered the section. This technique preserved the anatomical characteristics of the tissue, and provided spatial resolution sufficient for ready examination of protease activity in cells and in blood vessels within a single tissue section.