{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,7,2]],"date-time":"2025-07-02T15:00:43Z","timestamp":1751468443834,"version":"3.37.3"},"reference-count":14,"publisher":"Public Library of Science (PLoS)","issue":"5","license":[{"start":{"date-parts":[[2024,5,31]],"date-time":"2024-05-31T00:00:00Z","timestamp":1717113600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000001","name":"National Science Foundation","doi-asserted-by":"publisher","award":["IOS-1911723"],"award-info":[{"award-number":["IOS-1911723"]}],"id":[{"id":"10.13039\/100000001","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>The yellow fever mosquito <jats:italic>Aedes aegypti<\/jats:italic> is a major disease vector and an increasingly popular emerging model research organism. We present here an improved protocol for the collection, fixation, and preparation of <jats:italic>A. aegypti<\/jats:italic> embryos for immunohistochemical and <jats:italic>in situ<\/jats:italic> hybridization studies. The processing of <jats:italic>A. aegypti<\/jats:italic> embryos for such studies is complicated by the inability to easily remove the vitelline membrane, which prevents the reagents needed for staining from reaching their targets, and which furthermore obscures visualization of the embryo since the membrane is highly sclerotized. Previously described protocols for removal of the vitelline membrane are very low throughput, limiting the capacity of work that can be accomplished in a reasonable timeframe. Our adapted protocol increases the throughput capacity of embryos by an individual user, with experienced users able to prepare an average of 100\u2013150 embryos per hour. The protocol provides high-quality intact embryos that can be used for morphological, immunohistochemical, and <jats:italic>in situ<\/jats:italic> hybridization studies. The protocol has been successfully tested on embryos of ages ranging from 14h after egg laying (AEL) at 27\u00b0C through to 55h AEL. Critical to the success of the optimized protocol is the selection, fabrication, and description of the tools required. To this end, a video-demonstrated protocol has been placed at <jats:italic><jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" ext-link-type=\"uri\" xlink:href=\"https:\/\/protocols.io\" xlink:type=\"simple\">protocols.io<\/jats:ext-link><\/jats:italic> to clarify the protocol and provide easy access and training to anyone interested in the preparation of <jats:italic>A. aegypti<\/jats:italic> embryos for biological studies.<\/jats:p>","DOI":"10.1371\/journal.pone.0304802","type":"journal-article","created":{"date-parts":[[2024,5,31]],"date-time":"2024-05-31T19:21:36Z","timestamp":1717183296000},"page":"e0304802","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":2,"title":["Improved methodology for fixation and preparation of Aedes aegypti embryos"],"prefix":"10.1371","volume":"19","author":[{"given":"William","family":"Reid","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Geyenna","family":"Sterling-Lentsch","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4149-2705","authenticated-orcid":true,"given":"Marc S.","family":"Halfon","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"340","published-online":{"date-parts":[[2024,5,31]]},"reference":[{"key":"pone.0304802.ref001","doi-asserted-by":"crossref","first-page":"395","DOI":"10.1146\/annurev-med-050715-105122","article-title":"Zika, Chikungunya, and Other Emerging Vector-Borne Viral Diseases","volume":"69","author":"SC Weaver","year":"2018","journal-title":"Annu Rev Med"},{"key":"pone.0304802.ref002","doi-asserted-by":"crossref","first-page":"537","DOI":"10.1146\/annurev-ento-010814-020828","article-title":"Insecticide resistance in mosquitoes: impact, mechanisms, and research directions","volume":"60","author":"N. 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Conserved and novel enhancers in the Aedes aegypti single-minded locus recaptiulate embryonic ventral midline gene expression","volume":"20","author":"I Schember","year":"2024","journal-title":"PLoS Genetics"},{"issue":"1","key":"pone.0304802.ref014","doi-asserted-by":"crossref","first-page":"e16730","DOI":"10.1371\/journal.pone.0016730","article-title":"siRNA-mediated gene targeting in Aedes aegypti embryos reveals that frazzled regulates vector mosquito CNS development","volume":"6","author":"A Clemons","year":"2011","journal-title":"PLoS One"}],"container-title":["PLOS ONE"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0304802","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,5,31]],"date-time":"2024-05-31T19:21:45Z","timestamp":1717183305000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0304802"}},"subtitle":[],"editor":[{"given":"Ren\u00e9 Massimiliano","family":"Marsano","sequence":"first","affiliation":[],"role":[{"role":"editor","vocabulary":"crossref"}]}],"short-title":[],"issued":{"date-parts":[[2024,5,31]]},"references-count":14,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2024,5,31]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0304802","relation":{},"ISSN":["1932-6203"],"issn-type":[{"type":"electronic","value":"1932-6203"}],"subject":[],"published":{"date-parts":[[2024,5,31]]}}}