ArticleNature protocols2013
Optimized RNA ISH, RNA FISH and protein-RNA double labeling (IF/FISH) in Drosophila ovaries.
Article in Nature protocols, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 50 papers.
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Who cites it
50 citing papers in PubMed.
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- Commonly Overlooked Factors in Biocompatibility Studies of Neural Implants.Advanced science (Weinheim, Baden-Wurttemberg, Germany) · 2023Review
- Molecular characterization, expression, and function of Vitellogenin genes in Phytoseiulus persimilis.Experimental & applied acarology · 2022Article
- chinmo-mutant spermatogonial stem cells cause mitotic drive by evicting non-mutant neighbors from the niche.Developmental cell · 2022Article
Corrections and comments
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Authors and funding
4 authors.
Funding
Abstract
In situ hybridization (ISH) is a powerful technique for detecting nucleic acids in cells and tissues. Here we describe three ISH procedures that are optimized for Drosophila ovaries: whole-mount, digoxigenin-labeled RNA ISH; RNA fluorescent ISH (FISH); and protein immunofluorescence (IF)-RNA FISH double labeling (IF/FISH). Each procedure balances conflicting requirements for permeabilization, fixation and preservation of antigenicity to detect RNA and protein expression with high resolution and sensitivity. The ISH protocol uses alkaline phosphatase-conjugated digoxigenin antibodies followed by a color reaction, whereas FISH detection involves tyramide signal amplification (TSA). To simultaneously preserve antigens for protein detection and enable RNA probe penetration for IF/FISH, we perform IF before FISH and use xylenes and detergents to permeabilize the tissue rather than proteinase K, which can damage the antigens. ISH and FISH take 3 d to perform, whereas IF/FISH takes 5 d. Probe generation takes 1 or 2 d to perform.
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