Access to newer, fast and cheap sequencing techniques, particularly on the single-cell level, have made transcriptomic data of tissues or single cells accessible to many researchers. As a consequence, there is increased need for in situ visualization of gene expression or encoded proteins to validate, localize or help interpret such sequencing data, as well as put them in context with cellular proliferation. A particular challenge for labeling and imaging transcripts are complex tissues that are often opaque and/or pigmented, preventing easy visual inspection. Here we introduce a versatile protocol that combines in situ hybridization chain reaction (HCR), immunohistochemistry (IHC) and proliferative cell labeling using 5-ethynyl-2′-deoxyuridine (EdU), and demonstrate its compatibility with tissue clearing. As a proof-of-concept, we show that our protocol allows for the parallel analysis of cell proliferation, gene expression and protein localization in bristleworm heads and trunks.

Access to newer, fast and cheap sequencing techniques, particularly on the single-cell level, have made transcriptomic data of tissues or single cells accessible to many researchers. As a consequence, there is increased need for in situ visualization of gene expression or encoded proteins to validate, localize or help interpret such sequencing data, as well as put them in context with cellular proliferation. A particular challenge for labeling and imaging transcripts are complex tissues that are often opaque and/or pigmented, preventing easy visual inspection. Here we introduce a versatile protocol that combines in situ hybridization chain reaction (HCR), immunohistochemistry (IHC) and proliferative cell labeling using 5-ethynyl-2′-deoxyuridine (EdU), and demonstrate its compatibility with tissue clearing. As a proof-of-concept, we show that our protocol allows for the parallel analysis of cell proliferation, gene expression and protein localization in bristleworm heads and trunks.