{"database":"biostudies-literature","file_versions":[],"scores":null,"additional":{"submitter":["Zimmerman SG"],"funding":["NIGMS NIH HHS","PHS HHS"],"pagination":["2158-79"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/S-EPMC4126239"],"repository":["biostudies-literature"],"omics_type":["Unknown"],"volume":["8(11)"],"pubmed_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."],"journal":["Nature protocols"],"pubmed_title":["Optimized RNA ISH, RNA FISH and protein-RNA double labeling (IF/FISH) in Drosophila ovaries."],"pmcid":["PMC4126239"],"funding_grant_id":["R01 GM079433","T32 H600035","R01-GM079433"],"pubmed_authors":["Altaras AE","Zimmerman SG","Berg CA","Peters NC"],"additional_accession":[]},"is_claimable":false,"name":"Optimized RNA ISH, RNA FISH and protein-RNA double labeling (IF/FISH) in Drosophila ovaries.","description":"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.","dates":{"release":"2013-01-01T00:00:00Z","publication":"2013 Nov","modification":"2020-10-29T13:27:32Z","creation":"2019-03-27T01:33:44Z"},"accession":"S-EPMC4126239","cross_references":{"pubmed":["24113787"],"doi":["10.1038/nprot.2013.136"]}}