Multiplex immunohistochemistry assay to evaluate the melanoma tumor microenvironment.

The tumor microenvironment (TME) is composed of a set of cellular compartments comprising vascular, neuroendocrine, stromal, epithelial and immune cells. These compartments constitute a heterogeneous and dynamic set, where intercellular communication is driven by a complex network of cytokines, chemokines, growth factors, and inflammatory and matrix remodeling enzymes. Based on this complexity, an increasing number of assays may be required to identify and locate specific proteins in the tissue section and the standard procedure is to perform one stain at a time on serial sections. Recently, interest in performing multiple assays on formalin-fixed, paraffin embedded (FFPE) specimens has gained ground, and is referred to as multiplexing, i.e., multiple staining of the same section at the same time. Multiple staining is a promising approach that may help to improve understanding of the interactions between the different cellular components of the TME, stratify cancer patients, and help clinicians in their patient management. In this chapter, we detail a simple methodological approach to perform multiple staining on the same section using tissue obtained from patients with melanoma. This procedure evaluates the presence and location of three different proteins, human leukocyte antigen (HLA), forkhead box protein 3 (FoxP3) and Granzyme B (GRZB).