Multiparametric staining - combined application of immunofluorescence and in-situ hybridization

Immunohistochemistry (IHC) is a powerful technique that utilizes specific antibodies to visualize distinct cell populations within tissues. However, this method has some limitations, in particular the specificity and detection of low expressed markers including soluble factors, like e.g., cytokines. Other methods, such as in-situ hybridization (ISH), are a suitable alternative to visualize these factors in the tissue. Recent advances have opened exciting opportunities for quantitative data acquisition related to both protein and mRNA expression. Moreover, these techniques allow the analysis of their spatial distribution within a tissue and in a cell-specific context. Moreover, improvements in tissue preparation, fluorescent dyes, tissue imaging and downstream analysis have addressed challenges related to quantitative precision. Nowadays, researchers can obtain more accurately measurements of protein and mRNA expression levels of multiple targets within one sample. This technique gives us the opportunity to visualize and record the spatial relationship between different cells in formalin-fixed, paraffin-embedded samples. This chapter summarizes a protocol developed for cytokine expression analysis in the bone marrow of myeloid neoplasms. It provides an overview of the workflow that can be adapted to other tissues and other disease specific contexts.