Combined amplification-based single-molecule fluorescence in situ hybridization with immunofluorescence for simultaneous in situ detection of RNAs and proteins

We present a combined amplification-based single-molecule fluorescence in situ hybridization and immunofluorescence (asmFISH-IF) method for the detection of multiple RNAs and proteins simultaneously in cells and formaldehyde-fixed and paraffin-embedded tissue sections. We showed that performing asmFISH before immunofluorescence gives a better IF signal than the opposite. Our asmFISH-IF method could help study the interplay of RNA and protein, helping to understand their functions. •asmFISH-IF detects RNA and protein in situ simultaneously.•asmFISH-IF can be applied to both fixed cell and tissue sections.•asmFISH-IF offers an alternative option for protein-RNA co-detection.•Performing RNA detection before protein detection gives a better signal.