Biomarker discovery is a crucial part of the fast developing field of personalized medicine. Antibody-based techniques including immunostaining of tissue samples are widely used for biomarker evaluation in preclinical and clinical studies. When used in conjunction with robust image analysis methods, it provides a powerful means to assess biomarker modulation, toxicity, and patient response to targeted agents. Here, we describe the optimization of immunofluorescent (IF) staining protocols and a sample IF multiplex protocol suitable for colocalization image analysis.