Whole-Mount RNA FISH Combined with Immunofluorescence for the Analysis of the Telomeric Ribonucleoproteins in the Drosophila Germline

Abstract

The RNA fluorescence in situ hybridization (FISH) technique combined with immunostaining is a powerful method to visualize a specific transcript and a protein of interest simultaneously. Although whole-mount RNA FISH is routinely used to determine RNA intracellular localization, a detailed picture of RNA distribution in complex tissues remains a challenge. The main problem is the various permeability of morphologically different cells within a tissue. We overcome this challenge by developing an approach based on differential permeabilization treatment of tissue specimens. We have tested and optimized conditions for RNA FISH combined with immunofluorescent staining (RNA FISH/IF) to detect the maternal telomeric retrotransposon HeT-A RNPs in the Drosophila ovaries and syncytial embryos. Methods described here are applicable to a broad variety of biological tissue specimens.

Keywords: Combined RNA FISH and immunofluorescence; Drosophila; Embryogenesis; Germline; Oogenesis; Permeabilization; RNP; Retrotransposon HeT-A; Telomere; piRNA pathway.