Optimizing Tissue Preservation for High‐Resolution Confocal Imaging of Single‐Molecule RNA‐FISH

Abstract Over the past century, formalin‐fixed, paraffin‐embedded (FFPE) tissue samples have represented the standard for basic histology and immunostaining. However, FFPE has several limitations and less stringent tissue preservation methods are required for the visualization of nucleic acids at high resolution, particularly those that are expressed at low levels. Here, we describe the FFPE properties that negatively impact RNA integrity, an alternative tissue preservation technique that prevents RNA loss, and the steps necessary to optimize slide preparation for single‐molecule RNA fluorescent in situ hybridization (smRNA‐FISH) and imaging by confocal microscopy. This strategy retains RNA quality and eliminates formalin‐induced artifacts, thereby producing high‐resolution, diffraction‐limited confocal images of even rare RNA transcripts in tissues. As non‐coding RNAs and alternative splicing of gene isoforms continue to emerge as important regulators of human health and disease, a reliable, cost‐effective approach is required to examine the expression and localization of RNA targets in patient samples. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1 : Preparing an RNase‐free workstation Support Protocol 1 : Diethyl pyrocarbonate water treatment Support Protocol 2 : Removing RNase contamination from glassware Basic Protocol 2 : BE70 tissue fixation and processing Basic Protocol 3 : Cutting slide sections from paraffin blocks Basic Protocol 4 : Specimen pre‐treatment Basic Protocol 5 : RNA fluorescent in situ hybridization labeling Basic Protocol 6 : Slide mounting Basic Protocol 7 : Generating deconvolution‐capable confocal micrographs