{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,21]],"date-time":"2026-10-21T13:40:05Z","timestamp":1792590005629,"version":"4.5.0"},"reference-count":26,"publisher":"MyJove Corporation","issue":"231","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["JoVE"],"DOI":"10.3791\/71475","type":"journal-article","created":{"date-parts":[[2026,5,8]],"date-time":"2026-05-08T23:00:35Z","timestamp":1778281235000},"source":"Crossref","is-referenced-by-count":0,"title":["Multiplex Fluorescent mRNA &lt;em&gt;In Situ&lt;\/em&gt; Hybridization of Developing Craniofacial Tissues in FFPE Mouse Samples"],"prefix":"10.3791","author":[{"given":"Resmi","family":"Raju","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elena","family":"Makareeva","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marianna","family":"Bei","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"2098","published-online":{"date-parts":[[2026,5,8]]},"reference":[{"issue":"12","key":"ref1","doi-asserted-by":"crossref","first-page":"2387","DOI":"10.3390\/genes13122387","type":"journal-article","article-title":"Real-time polymerase chain reaction: current techniques, applications, and role in COVID-19 diagnosis","volume":"13","author":"Artika","year":"2022","unstructured":"Artika, I. M. et al. Real-time polymerase chain reaction: current techniques, applications, and role in COVID-19 diagnosis. Genes. 13 (12), 2387 (2022).","journal-title":"Genes"},{"issue":"5","key":"ref2","doi-asserted-by":"crossref","first-page":"336","DOI":"10.1053\/j.semdp.2019.06.004","type":"journal-article","article-title":"In situ hybridization: introduction to techniques, applications and pitfalls in the performance and interpretation of assays","volume":"36","author":"Chu","year":"2019","unstructured":"Chu, Y. H. et al. In situ hybridization: introduction to techniques, applications and pitfalls in the performance and interpretation of assays. Semin Diagn Pathol. 36 (5), 336\u2013341 (2019).","journal-title":"Semin Diagn Pathol"},{"issue":"1","key":"ref3","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1007\/s40291-021-00570-2","type":"journal-article","article-title":"Evaluation of the suitability of RNAscope as a technique to measure gene expression in clinical diagnostics: a systematic review","volume":"26","author":"Atout","year":"2022","unstructured":"Atout, S. et al. Evaluation of the suitability of RNAscope as a technique to measure gene expression in clinical diagnostics: a systematic review. Mol Diagn Ther. 26 (1), 19\u201337 (2022).","journal-title":"Mol Diagn Ther"},{"issue":"19","key":"ref4","doi-asserted-by":"crossref","first-page":"dev202077","DOI":"10.1242\/dev.202077","type":"journal-article","article-title":"Craniofacial developmental biology in the single-cell era","volume":"150","author":"Tseng","year":"2023","unstructured":"Tseng, K. C., Crump, J. G. Craniofacial developmental biology in the single-cell era. Development. 150 (19), dev202077 (2023).","journal-title":"Development"},{"issue":"6","key":"ref5","doi-asserted-by":"crossref","first-page":"533","DOI":"10.1038\/s41592-019-0404-0","type":"journal-article","article-title":"SABER amplifies FISH: enhanced multiplexed imaging of RNA and DNA in cells and tissues","volume":"16","author":"Kishi","year":"2019","unstructured":"Kishi, J. Y. et al. SABER amplifies FISH: enhanced multiplexed imaging of RNA and DNA in cells and tissues. Nat Methods. 16 (6), 533\u2013544 (2019).","journal-title":"Nat Methods"},{"key":"ref6","doi-asserted-by":"crossref","first-page":"109","DOI":"10.1007\/978-1-0716-2659-7_6","type":"journal-article","article-title":"Hybridization chain reaction fluorescence in situ hybridization (HCR-FISH) in Ambystoma mexicanum tissue","volume":"2562","author":"Lovely","year":"2023","unstructured":"Lovely, A. M. et al. Hybridization chain reaction fluorescence in situ hybridization (HCR-FISH) in Ambystoma mexicanum tissue. Methods Mol Biol. 2562, 109\u2013122 (2023).","journal-title":"Methods Mol Biol"},{"key":"ref7","doi-asserted-by":"crossref","first-page":"1316635","DOI":"10.3389\/fphys.2023.1316635","type":"journal-article","article-title":"Profiles of Wnt pathway gene expression during tooth morphogenesis","volume":"14","author":"Raju","year":"2024","unstructured":"Raju, R. et al. Profiles of Wnt pathway gene expression during tooth morphogenesis. Front Physiol. 14, 1316635 (2024).","journal-title":"Front Physiol"},{"key":"ref8","doi-asserted-by":"crossref","first-page":"5687","DOI":"10.1038\/s41467-023-41349-9","type":"journal-article","article-title":"Multimodal spatiotemporal transcriptomic resolution of embryonic palate osteogenesis","volume":"14","author":"Pi\u00f1a","year":"2023","unstructured":"Pi\u00f1a, J. O. et al. Multimodal spatiotemporal transcriptomic resolution of embryonic palate osteogenesis. Nat Commun. 14, 5687 (2023).","journal-title":"Nat Commun"},{"issue":"13","key":"ref9","doi-asserted-by":"crossref","first-page":"1412","DOI":"10.1177\/00220345241256600","type":"journal-article","article-title":"Spatial multi-omics reveals the role of the Wnt modulator Dkk2 in palatogenesis","volume":"103","author":"Pi\u00f1a","year":"2024","unstructured":"Pi\u00f1a, J. O. et al. Spatial multi-omics reveals the role of the Wnt modulator Dkk2 in palatogenesis. J Dent Res. 103 (13), 1412\u20131420 (2024).","journal-title":"J Dent Res"},{"issue":"10","key":"ref10","doi-asserted-by":"crossref","first-page":"877","DOI":"10.1038\/nmeth.1253","type":"journal-article","article-title":"Imaging individual mRNA molecules using multiple singly labeled probes","volume":"5","author":"Raj","year":"2008","unstructured":"Raj, A. et al. Imaging individual mRNA molecules using multiple singly labeled probes. Nat Methods. 5 (10), 877\u2013879 (2008).","journal-title":"Nat Methods"},{"key":"ref11","doi-asserted-by":"crossref","first-page":"19490","DOI":"10.1073\/pnas.1912459116","type":"journal-article","article-title":"Spatial transcriptome profiling by MERFISH reveals subcellular RNA compartmentalization and cell cycle-dependent gene expression","volume":"116","author":"Xia","year":"2019","unstructured":"Xia, C. et al. Spatial transcriptome profiling by MERFISH reveals subcellular RNA compartmentalization and cell cycle-dependent gene expression. Proc Natl Acad Sci USA. 116, 19490\u201319499 (2019).","journal-title":"Proc Natl Acad Sci USA"},{"key":"ref12","doi-asserted-by":"crossref","first-page":"360","DOI":"10.1038\/nmeth.2892","type":"journal-article","article-title":"Single-cell in situ RNA profiling by sequential hybridization","volume":"11","author":"Lubeck","year":"2014","unstructured":"Lubeck, E. et al. Single-cell in situ RNA profiling by sequential hybridization. Nat Methods. 11, 360\u2013361 (2014).","journal-title":"Nat Methods"},{"key":"ref13","doi-asserted-by":"crossref","first-page":"342","DOI":"10.1016\/j.neuron.2016.10.001","type":"journal-article","article-title":"In situ transcription profiling of single cells reveals spatial organization of cells in the mouse hippocampus","volume":"92","author":"Shah","year":"2016","unstructured":"Shah, S. et al. In situ transcription profiling of single cells reveals spatial organization of cells in the mouse hippocampus. Neuron. 92, 342\u2013357 (2016).","journal-title":"Neuron"},{"issue":"2","key":"ref14","doi-asserted-by":"crossref","first-page":"135","DOI":"10.1002\/wdev.170","type":"journal-article","article-title":"Strength in numbers: quantitative single-molecule RNA detection assays","volume":"4","author":"Gaspar","year":"2015","unstructured":"Gaspar, I., Ephrussi, A. Strength in numbers: quantitative single-molecule RNA detection assays. Wiley Interdiscip Rev Dev Biol. 4 (2), 135\u2013150 (2015).","journal-title":"Wiley Interdiscip Rev Dev Biol"},{"key":"ref15","doi-asserted-by":"crossref","first-page":"e63","DOI":"10.1002\/cpns.63","type":"journal-article","article-title":"Detection and quantification of multiple RNA sequences using emerging ultrasensitive fluorescent in situ hybridization techniques","volume":"87","author":"Erben","year":"2019","unstructured":"Erben, L., Buonanno, A. Detection and quantification of multiple RNA sequences using emerging ultrasensitive fluorescent in situ hybridization techniques. Curr Protoc Neurosci. 87 ( ), e63 (2019).","journal-title":"Curr Protoc Neurosci"},{"issue":"2","key":"ref16","doi-asserted-by":"crossref","first-page":"177","DOI":"10.1093\/jbmr\/zjad004","type":"journal-article","article-title":"RNA-based bone histomorphometry: method and its application","volume":"39","author":"Makareeva","year":"2024","unstructured":"Makareeva, E. et al. RNA-based bone histomorphometry: method and its application. J Bone Miner Res. 39 (2), 177\u2013189 (2024).","journal-title":"J Bone Miner Res"},{"issue":"1","key":"ref17","doi-asserted-by":"crossref","first-page":"22","DOI":"10.1016\/j.jmoldx.2011.08.002","type":"journal-article","article-title":"RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues","volume":"14","author":"Wang","year":"2012","unstructured":"Wang, F. et al. RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. J Mol Diagn. 14 (1), 22\u201329 (2012).","journal-title":"J Mol Diagn"},{"issue":"3","key":"ref18","doi-asserted-by":"crossref","first-page":"92","DOI":"10.1177\/01926233231178282","type":"journal-article","article-title":"Review of in situ hybridization techniques for drug research and development","volume":"51","author":"Monn\u00e9 Rodr\u00edguez","year":"2023","unstructured":"Monn\u00e9 Rodr\u00edguez, J. M. et al. Review of in situ hybridization techniques for drug research and development. Toxicol Pathol. 51 (3), 92\u2013111 (2023).","journal-title":"Toxicol Pathol"},{"issue":"4","key":"ref19","doi-asserted-by":"crossref","first-page":"498","DOI":"10.1177\/10406387241245777","type":"journal-article","article-title":"Effect of formalin-fixation and paraffin-embedded tissue storage times on RNAscope signal amplification","volume":"36","author":"Colburn","year":"2024","unstructured":"Colburn, M. E. et al. Effect of formalin-fixation and paraffin-embedded tissue storage times on RNAscope signal amplification. J Vet Diagn Invest. 36 (4), 498\u2013505 (2024).","journal-title":"J Vet Diagn Invest"},{"issue":"3","key":"ref20","doi-asserted-by":"crossref","first-page":"429","DOI":"10.1309\/AJCPDZ4FAQ9BUEXC","type":"journal-article","article-title":"Validation of a postfixation tissue storage and transport medium","volume":"137","author":"Stumm","year":"2012","unstructured":"Stumm, M. M. et al. Validation of a postfixation tissue storage and transport medium. Am J Clin Pathol. 137 (3), 429\u2013436 (2012).","journal-title":"Am J Clin Pathol"},{"key":"ref21","volume-title":"RNAscope multiplex fluorescent reagent kit v2 user manual","year":"2025","unstructured":"ACDbio. RNAscope multiplex fluorescent reagent kit v2 user manual. At < https:\/\/manuals.plus\/m\/0f24eea864dcf70b83735b261589c2e98f9c707b01c1c9c0455f349c8ab2eb0c> (2025).","type":"book"},{"key":"ref22","doi-asserted-by":"crossref","first-page":"16878","DOI":"10.1038\/s41598-017-17204-5","type":"journal-article","article-title":"QuPath: open source software for digital pathology image analysis","volume":"7","author":"Bankhead","year":"2017","unstructured":"Bankhead, P. et al. QuPath: open source software for digital pathology image analysis. Sci Rep. 7, 16878 (2017).","journal-title":"Sci Rep"},{"key":"ref23","doi-asserted-by":"crossref","first-page":"216","DOI":"10.1038\/s41597-025-06533-0","type":"journal-article","article-title":"Single-cell and spatial transcriptomic profiling of cardiac fibroblasts following myocardial infarction","volume":"13","author":"Hern\u00e1ndez","year":"2026","unstructured":"Hern\u00e1ndez, S. C. et al. Single-cell and spatial transcriptomic profiling of cardiac fibroblasts following myocardial infarction. Sci Data. 13, 216 (2026).","journal-title":"Sci Data"},{"key":"ref24","doi-asserted-by":"crossref","first-page":"150409","DOI":"10.1016\/j.bbrc.2024.150409","type":"journal-article","article-title":"The TGF-\u03b21 target WISP1 is highly expressed in liver cirrhosis and cirrhotic HCC microenvironment","volume":"732","author":"Dropmann","year":"2024","unstructured":"Dropmann, A. et al. The TGF-\u03b21 target WISP1 is highly expressed in liver cirrhosis and cirrhotic HCC microenvironment. Biochem Biophys Res Commun. 732, 150409 (2024).","journal-title":"Biochem Biophys Res Commun"},{"issue":"224","key":"ref25","first-page":"e68615","type":"journal-article","article-title":"RNA in situ hybridization with sequential protein immunofluorescence in tandem assay","author":"Stolley","year":"2025","unstructured":"Stolley, D. L. et al. RNA in situ hybridization with sequential protein immunofluorescence in tandem assay. J Vis Exp. 224, e68615 (2025).","journal-title":"J Vis Exp"},{"issue":"138","key":"ref26","first-page":"e58097","type":"journal-article","article-title":"Visualizing genetic variants, short targets, and point mutations in the morphological tissue context","author":"Anderson","year":"2018","unstructured":"Anderson, C. M. et al. Visualizing genetic variants, short targets, and point mutations in the morphological tissue context. J Vis Exp. 138, e58097 (2018).","journal-title":"J Vis Exp"}],"container-title":["Journal of Visualized Experiments"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.jove.com\/t\/71475\/multiplex-fluorescent-mrna-situ-hybridization-developing-craniofacial","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,21]],"date-time":"2026-10-21T12:50:48Z","timestamp":1792587048000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.jove.com\/t\/71475\/multiplex-fluorescent-mrna-situ-hybridization-developing-craniofacial"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,5,8]]},"references-count":26,"journal-issue":{"issue":"231","published-online":{"date-parts":[[2026]]}},"URL":"https:\/\/doi.org\/10.3791\/71475","relation":{},"ISSN":["1940-087X"],"issn-type":[{"value":"1940-087X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,5,8]]}}}