Immunohistochemical amplification of mCherry fusion protein is necessary for proper visualization

MethodsX. 2020 Jun 2;7:100946. doi: 10.1016/j.mex.2020.100946. eCollection 2020.


Fluorescent reporter proteins are a powerful tool being increasingly integrated into biological experiments. Their utility spans techniques such as live-cell imaging, validating transgene expression, and studying cell-type specific anatomy. As these reporters become more widely used, it is necessary to fully understand their benefits and limitations. One such recently developed red fluorescent protein, mCherry, has been well utilized due to its stability, brightness, and pH resistance. In the course of an experiment using the fluorescent reporter protein mCherry fused to a G-protein coupled receptor (mCherry fusion protein), our lab discovered a notable inability for the fusion protein to faithfully produce fluorescent signal representative of its expression in fixed tissue. Here, we demonstrate the importance of immunohistochemical amplification in tissue injected with various adeno-associated viruses (AAVs), containing mCherry fusion protein as a reporter. Our findings demonstrate that antibody amplification consistently provides a stronger signal when mCherry fusion protein is used as a reporter protein.

Keywords: Adeno-associated virus; DREADD; Fluorescence microscopy; Immunohistochemistry; Reporter protein; mCherry fusion protein.