Robust RNAScope® Assay for RNA and Protein Co-Detection (p21 probe)

We used the RNAScope® Multiplex Fluorescent V2 Assay to detect p21 (Rn-Cdkn1a, #42851, ACD) in formalin-fixed, paraffin-embedded (FFPE) brain samples from young and aged rats. Although the assay is robust and reproducible, it involves numerous hybridization steps. When combined with immunofluorescence staining for additional protein targets (e.g., VE-cadherin), the complete protocol required approximately three days. The assay also required optimization for different tissue types and microscopy settings. Once these parameters were optimized, the protocol proved to be reliable, robust, and reproducible.