Detection of antigen is dependent on the expression level in tissue, fixation, antibody avidity, and specificity in combination with detection systems. Detection using primary antibodies raised in the same species is often complicated and may reduce the choice of antibodies used. In the present chapter, a protocol for multicolor immunohistochemical staining of brain tissue containing the suprachiasmatic nucleus is presented in which the use of two antibodies raised in the same species in combination with antibodies raised in other species. The primary antibodies are visualized using amplification steps including biotinylated tyramide and amplification using the EnVision-based system and tyramide and classical indirect immunohistochemistry to enhance signal to noise in order to perform confocal microscopy. Confocal microscopy using a spinning disk system creates stacks of multicolor images, which can be stitched, reconstructed in 3D, and analyzed.