Detecting Reactive Oxygen Species by Immunohistochemistry

In cultured cells, an increase in cellular levels of reactive oxygen species (ROS) can be detected using multiple techniques including colorimetric assays, immunoblotting, and immunofluorescence. These methods can also be applied for ROS measurement in tissue samples, but often require tissue homogenization, and therefore do not distinguish between the different cell types within a tissue. Here, we describe a detailed protocol for determination of altered oxidative stress levels in different cell types in tissues, by detecting ROS-caused alteration of macromolecules using immunohistochemistry (IHC). This method is demonstrated by using 4HNE as a marker for lipid peroxidation in mouse pancreas tissue that contains precancerous lesions high in cellular oxidative stress.