Whole-Mount RNA FISH Combined with Immunofluorescence for the Analysis of the Telomeric Ribonucleoproteins in the Drosophila Germline.
Methods in molecular biology (Clifton, N.J.)(2022)
摘要
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.
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关键词
Combined RNA FISH and immunofluorescence,Drosophila,Embryogenesis,Germline,Oogenesis,Permeabilization,RNP,Retrotransposon HeT-A,Telomere,piRNA pathway
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