Whole Mount Fluorescent In situ Hybridization of miRNA Combined with Immunofluorescence of Proteins in Intact Islets

Abstract

Pancreatic islets primarily involved in regulating blood glucose homeostasis are the key focus of diabetes study and miRNA-mediated molecular regulation. Whole mount in situ hybridization of miRNAs and co-localization of proteins is a powerful tool for studying cell-specific expression and regulation. Although many miRNAs are potential regulators of islet function and diabetes development, most of their cell specific expression remains unknown. We developed and optimized a protocol that combines fluorescent in situ hybridization (FISH) of miRNAs and immunofluorescence (IF) of two proteins at the same time in whole mount isolated intact islets for preserving islet morphology, eliminating the technical difficulties associated with handling isolated islets that are not amenable to conventional FISH, as well as for increasing sensitivity and resolution. This method can serve as a valuable tool for studying miRNA-mediated regulation of post-transcriptional gene expression in islet biology, diabetes research, and biomarker development.