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Fluorescent reporters have been widely used in modern biology as a powerful tool in cell lineage tracing during development and in studying the pathogenesis of diseases. RNAscope is a recently developed RNA in situ hybridization method with high specificity and sensitivity. Combined application of these two techniques on skeletal tissue is technically challenging and has not been explored; the reporter fluorophores in the tissue specimen bleach quickly, and mRNAs degrade rapidly due to the decalcification process typically used in processing skeletal samples. Therefore, we developed a method that can simultaneously detect and colocalize both the fluorescent lineage tracing reporter signal and the RNAscope signal in the same skeletal section without compromising the fidelity, sensitivity, and specificity of lineage tracing and RNAscope. This was achieved by cryosectioning bone and cartilage tissue without decalcification, thus allowing the fluorescent reporter signal and RNA in the sections to be well-preserved so that RNAscope can be carried out in situ, and these two signals can be colocalized. Our method of colocalization has versatile applications, for example, determination of gene knockout efficacy at the mRNA level in a specific cell lineage in situ, detection of alterations in target gene transcripts in reporter-positive cells caused by a specific gene mutation, and studies of the disease pathology by examining the transcript-level expression of genes of interest in the cell lineage in vivo.

A method for colocalizing lineage tracing reporter and RNAscope signals on skeletal tissue section