{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,16]],"date-time":"2026-05-16T04:58:55Z","timestamp":1778907535891,"version":"3.51.4"},"reference-count":26,"publisher":"Wiley","issue":"6","license":[{"start":{"date-parts":[[2023,6,22]],"date-time":"2023-06-22T00:00:00Z","timestamp":1687392000000},"content-version":"vor","delay-in-days":21,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Current Protocols"],"published-print":{"date-parts":[[2023,6]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p><jats:italic>In situ<\/jats:italic> hybridization (ISH) is a sensitive method used to localize a specific sequence of DNA or RNA in biological samples, including cells, tissue sections or whole organs. RNA ISH can be used to determine spatial gene expression using a single\u2010stranded probe with a reverse\u2010complementary sequence. Cell\u2010specific gene expression has been studied using mRNA and protein levels. Signals produced by RNA probes are usually more specific than those produced by antibodies in immunostaining. Currently, ISH is the most widely used method to localize mRNA molecules. Traditionally, probes were labeled with radioactive isotopes, but the cumbersome procedures and potential health risk limit their acceptance. Recently, probes labeled with nonradioactive materials including digoxigenin, biotin and various fluorophores have been developed. The tyramide signal amplification system further enhances the sensitivity of detection. These methods have been applied in numerous studies in various tissues including reproductive organs. This article details three methods of RNA <jats:italic>in situ<\/jats:italic> hybridization: radioactive <jats:italic>in situ<\/jats:italic> hybridization, digoxigenin <jats:italic>in situ<\/jats:italic> hybridization, and digoxigenin\u2010tyramide signal amplification fluorescein <jats:italic>in situ<\/jats:italic> hybridization. The pros and cons of each protocol are discussed. \u00a9 2023 Wiley Periodicals LLC.<\/jats:p><jats:p><jats:bold>Basic Protocol 1<\/jats:bold>: Radioactive <jats:italic>in situ<\/jats:italic> hybridization (radioactive\u2010ISH)<\/jats:p><jats:p><jats:bold>Basic Protocol 2<\/jats:bold>: Digoxigenin <jats:italic>in situ<\/jats:italic> hybridization (DIG\u2010ISH)<\/jats:p><jats:p><jats:bold>Basic Protocol 3<\/jats:bold>: Digoxigenin\u2010tyramide signal amplification fluorescein <jats:italic>in situ<\/jats:italic> hybridization (DIG\u2010TSA\u2010FISH)<\/jats:p>","DOI":"10.1002\/cpz1.823","type":"journal-article","created":{"date-parts":[[2023,6,24]],"date-time":"2023-06-24T20:29:34Z","timestamp":1687638574000},"source":"Crossref","is-referenced-by-count":4,"title":["Tissue\u2010Specific RNA Localization in the Uterus During Implantation, Decidualization and Placentation: Technical Nuances of Various Labeling Approaches"],"prefix":"10.1002","volume":"3","author":[{"given":"Amanda","family":"Dewar","sequence":"first","affiliation":[{"name":"Division of Developmental Biology Cincinnati Children's Hospital Medical Center  Cincinnati Ohio"},{"name":"College of Medicine University of Cincinnati  Cincinnati Ohio"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wenbo","family":"Deng","sequence":"additional","affiliation":[{"name":"Division of Developmental Biology Cincinnati Children's Hospital Medical Center  Cincinnati Ohio"},{"name":"College of Medicine University of Cincinnati  Cincinnati Ohio"},{"name":"Present affiliation: Fujian Provincial Key Laboratory of Reproductive Health Research, School of Medicine Xiamen University  Xiamen Fujian 361009 China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xiaofei","family":"Sun","sequence":"additional","affiliation":[{"name":"Division of Developmental Biology Cincinnati Children's Hospital Medical Center  Cincinnati Ohio"},{"name":"College of Medicine University of Cincinnati  Cincinnati Ohio"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sudhansu K.","family":"Dey","sequence":"additional","affiliation":[{"name":"Division of Developmental Biology Cincinnati Children's Hospital Medical Center  Cincinnati Ohio"},{"name":"College of Medicine University of Cincinnati  Cincinnati Ohio"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2023,6,22]]},"reference":[{"key":"e_1_2_9_2_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41418\u2010019\u20100429\u2010z"},{"key":"e_1_2_9_3_1","doi-asserted-by":"publisher","DOI":"10.1002\/cpmo.74"},{"key":"e_1_2_9_4_1","doi-asserted-by":"publisher","DOI":"10.1002\/0471143030.cb2204s23"},{"key":"e_1_2_9_5_1","doi-asserted-by":"publisher","DOI":"10.1016\/s1534\u20105807(02)00365\u20109"},{"key":"e_1_2_9_6_1","doi-asserted-by":"publisher","DOI":"10.1177\/030098589803500301"},{"key":"e_1_2_9_7_1","doi-asserted-by":"publisher","DOI":"10.1002\/9780471729259.mca02as00"},{"key":"e_1_2_9_8_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.63.2.378"},{"key":"e_1_2_9_9_1","doi-asserted-by":"publisher","DOI":"10.1385\/1\u201059745\u2010229\u20107:27"},{"key":"e_1_2_9_10_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/18.19.5843"},{"key":"e_1_2_9_11_1","doi-asserted-by":"publisher","DOI":"10.1038\/223582a0"},{"key":"e_1_2_9_12_1","doi-asserted-by":"publisher","DOI":"10.1210\/endo.140.6.6716"},{"key":"e_1_2_9_13_1","doi-asserted-by":"publisher","DOI":"10.1097\/00019606-199206000-00008"},{"key":"e_1_2_9_14_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.78.11.6633"},{"issue":"4","key":"e_1_2_9_15_1","first-page":"604","article-title":"Synthesis 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