The immunohistological methods described in this unit can be used to determine the precise localization of neurochemicals, receptors, and proteins throughout the nervous system. Determining the localization of a protein within defined brain nuclei and neuronal cell populations can provide important clues regarding its potential function. Immunoperoxidase reactions and light microscopy are commonly used to visualize the distribution of a single primary antibody directed to an antigen of interest. Double-labeling immunofluorescence and confocal microscopy techniques detect the localization of one protein relative to another protein and allow analysis of colocalization at a cellular and subcellular level. The colocalization of two proteins can also be quantified, allowing analysis of the extent of overlap between two labeled markers and measurements of changes in the localization of one protein relative to another following drug treatment or in animals that have been genetically modified. The theoretical limit of resolution of confocal microscopy is 0.1 to 0.2 microm.