This technical note provides guidelines for performing automated fluorescent co-detection of RNA and protein by combining in situ hybridization (ISH) using an RNAscope LS Multiplex Fluorescent Reagent Kit (Cat. No. 322800) and immunofluorescence (IF) on the Leica BOND RX System.
You can use these protocols with a wide range of protein targets, including those that are protease sensitive. On the Leica BOND RX, the samples undergo deparaffinization and heat-induced epitope retrieval, followed by primary antibody incubation and post-primary fixation. Samples are then treated with protease and undergo probe hybridization and full LS Multiplex RNAscope detection, followed by secondary antibody incubation, detection, and counterstaining.
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