**Abstract**

During the last years, multiplex immunofluorescence (mIF) has emerged as a very powerful tool in multiple epitope detection to study tumor tissues. This revolutionary technology is providing an important visual technique for tumor examination in formalin-fixed paraffin-embedded specimens for a better understanding of tumor microenvironment, new treatment discoveries, cancer prevention, as well as translational studies. The aim of this chapter is to highlight the use of tyramide signal amplification methodology in mIF and image analysis to identify several proteins at the same time in one single tissue and their spatial distribution in different tumor specimens including whole sections, core needle biopsies, and tissue microarrays. This type of methodology associated with image analysis can perform high-quality throughput assay in translational research studies to be applied in cancer prevention and treatments.

**Keywords:** tyramide signal amplification, conventional IHC protocol, immunoprofiling, cancer tissues, image analysis, spatial analysis
