In situ hybridization (ISH) techniques have been important to the study of gene expression signatures in cells and tissues. The ability to detect multiple targets simultaneously is especially valuable, since it allows dissecting gene expression of distinct cell types with precise cellular and subcellular resolution within morphological context. Recently, we have reported using a novel dual-color ultrasensitive bright-field RNA in situ hybridization for detection of clonally restricted immunoglobulin light chain mRNA expression in B cell lymphomas. Here, we present detailed protocols of RNAscope 2-Plex assays for FFPE tissue sections. The protocols describe the tissue preparation, pretreatment, probe hybridization, signal amplification, visualization, and analysis, as well as emphasize the critical steps for ensuring successful staining.