HiPlex RNAscope™ for In Situ Analysis of Gene Expression During Murine Thymocyte Differentiation.

RNAscope is a technique for performing multiplexed in situ hybridization (ISH) to localize and quantify specific RNA molecules within cells or tissues. We were interested in exploring the technique for thymocyte differentiation and migration in the complex process of T-cell development. In the thymus, thymocytes undergo various stages of differentiation, selection, and maturation as they migrate through the thymic cortex and medulla. Direct interactions within the different thymic microenvironments (the cortical or medullary stroma) ensure that thymocytes experience developmental events at the right place and in the right temporal order. These stages are critical to the correct functioning of T cells and allow them to become both MHC restricted and self-tolerant. The expanded range of the RNAscope HiPlex platform allows fluorescent, multiplexed, and quantitative analysis of the expression of up to 12 different genes in formalin-fixed paraffin-embedded (FFPE) sections. Comparing gene expression patterns of thymocyte differentiation and maturation markers will enable us to follow the migration and development of the thymocytes through thymic cortex and medulla. With this information, we can now simultaneously image the expression and location of our genes of biological interest within the thymic compartments and map them to different stages of thymocytes differentiation. A detailed protocol is provided for whole section preparation of HiPlex RNAscope of the murine thymus.