{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,16]],"date-time":"2026-04-16T20:36:41Z","timestamp":1776371801478,"version":"3.51.2"},"reference-count":12,"publisher":"Wiley","issue":"1","license":[{"start":{"date-parts":[[2019,10,16]],"date-time":"2019-10-16T00:00:00Z","timestamp":1571184000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":["currentprotocols.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["CP Molecular Biology"],"published-print":{"date-parts":[[2019,12]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Over the past century, formalin\u2010fixed, paraffin\u2010embedded (FFPE) tissue samples have represented the standard for basic histology and immunostaining. However, FFPE has several limitations and less stringent tissue preservation methods are required for the visualization of nucleic acids at high resolution, particularly those that are expressed at low levels. Here, we describe the FFPE properties that negatively impact RNA integrity, an alternative tissue preservation technique that prevents RNA loss, and the steps necessary to optimize slide preparation for single\u2010molecule RNA fluorescent in situ hybridization (smRNA\u2010FISH) and imaging by confocal microscopy. This strategy retains RNA quality and eliminates formalin\u2010induced artifacts, thereby producing high\u2010resolution, diffraction\u2010limited confocal images of even rare RNA transcripts in tissues. As non\u2010coding RNAs and alternative splicing of gene isoforms continue to emerge as important regulators of human health and disease, a reliable, cost\u2010effective approach is required to examine the expression and localization of RNA targets in patient samples. \u00a9 2019 by John Wiley &amp; Sons, Inc.<\/jats:p><jats:p><jats:bold>Basic Protocol 1<\/jats:bold>: Preparing an RNase\u2010free workstation<\/jats:p><jats:p><jats:bold>Support Protocol 1<\/jats:bold>: Diethyl pyrocarbonate water treatment<\/jats:p><jats:p><jats:bold>Support Protocol 2<\/jats:bold>: Removing RNase contamination from glassware<\/jats:p><jats:p><jats:bold>Basic Protocol 2<\/jats:bold>: BE70 tissue fixation and processing<\/jats:p><jats:p><jats:bold>Basic Protocol 3<\/jats:bold>: Cutting slide sections from paraffin blocks<\/jats:p><jats:p><jats:bold>Basic Protocol 4<\/jats:bold>: Specimen pre\u2010treatment<\/jats:p><jats:p><jats:bold>Basic Protocol 5<\/jats:bold>: RNA fluorescent in situ hybridization labeling<\/jats:p><jats:p><jats:bold>Basic Protocol 6<\/jats:bold>: Slide mounting<\/jats:p><jats:p><jats:bold>Basic Protocol 7<\/jats:bold>: Generating deconvolution\u2010capable confocal micrographs<\/jats:p>","DOI":"10.1002\/cpmb.107","type":"journal-article","created":{"date-parts":[[2019,10,16]],"date-time":"2019-10-16T22:27:57Z","timestamp":1571264877000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Optimizing Tissue Preservation for High\u2010Resolution Confocal Imaging of Single\u2010Molecule RNA\u2010FISH"],"prefix":"10.1002","volume":"129","author":[{"given":"Nash","family":"Redmayne","sequence":"first","affiliation":[{"name":"Division of Reproductive &amp; Developmental Sciences Oregon National Primate Research Center  Beaverton Oregon"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shawn L.","family":"Chavez","sequence":"additional","affiliation":[{"name":"Division of Reproductive &amp; Developmental Sciences Oregon National Primate Research Center  Beaverton Oregon"},{"name":"Department of Obstetrics &amp; Gynecology Oregon Health &amp; Science University School of Medicine  Portland Oregon"},{"name":"Department of Physiology &amp; Pharmacology Oregon Health &amp; Science University School of Medicine  Portland Oregon"},{"name":"Department of Biomedical Engineering Oregon Health &amp; Science University School of Medicine  Portland Oregon"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2019,10,16]]},"reference":[{"key":"e_1_2_13_2_1_1","doi-asserted-by":"publisher","DOI":"10.1021\/nn405717p"},{"key":"e_1_2_13_2_2_1","doi-asserted-by":"publisher","DOI":"10.1038\/nbt.1692"},{"key":"e_1_2_13_2_3_1","doi-asserted-by":"publisher","DOI":"10.1242\/dev.165753"},{"key":"e_1_2_13_2_4_1","doi-asserted-by":"publisher","DOI":"10.1369\/0022155417741467"},{"key":"e_1_2_13_2_5_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0002\u20109440(10)63093\u20103"},{"key":"e_1_2_13_2_6_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmoldx.2011.01.010"},{"key":"e_1_2_13_2_7_1","doi-asserted-by":"publisher","DOI":"10.1101\/pdb.top101857"},{"key":"e_1_2_13_2_8_1","doi-asserted-by":"publisher","DOI":"10.1002\/0471142727.mb1415s102"},{"key":"e_1_2_13_2_9_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.R115.651679"},{"key":"e_1_2_13_2_10_1","doi-asserted-by":"publisher","DOI":"10.1369\/0022155416649579"},{"key":"e_1_2_13_3_2_1","unstructured":"https:\/\/www.thermofisher.com\/us\/en\/home\/life\u2010science\/cell\u2010analysis\/labeling\u2010chemistry\/fluorescence\u2010spectraviewer.html"},{"key":"e_1_2_13_3_4_1","unstructured":"https:\/\/imagej.net\/Deconvolution"}],"container-title":["Current Protocols in Molecular Biology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cpmb.107","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/full-xml\/10.1002\/cpmb.107","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cpmb.107","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,8,22]],"date-time":"2023-08-22T15:13:27Z","timestamp":1692717207000},"score":1,"resource":{"primary":{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/10.1002\/cpmb.107"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2019,10,16]]},"references-count":12,"journal-issue":{"issue":"1","published-print":{"date-parts":[[2019,12]]}},"alternative-id":["10.1002\/cpmb.107"],"URL":"https:\/\/doi.org\/10.1002\/cpmb.107","archive":["Portico"],"relation":{},"ISSN":["1934-3639","1934-3647"],"issn-type":[{"value":"1934-3639","type":"print"},{"value":"1934-3647","type":"electronic"}],"subject":[],"published":{"date-parts":[[2019,10,16]]},"assertion":[{"value":"2019-10-16","order":2,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e107"}}