{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,7,30]],"date-time":"2025-07-30T09:45:17Z","timestamp":1753868717470,"version":"3.41.2"},"reference-count":61,"publisher":"Wiley","issue":"12","license":[{"start":{"date-parts":[[2021,12,22]],"date-time":"2021-12-22T00:00:00Z","timestamp":1640131200000},"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":[[2021,12]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Zika virus is an arthropod\u2010borne virus that has recently emerged as a significant public health emergency due to its association with congenital malformations. Serological and molecular tests are typically used to confirm Zika virus infection. These methods, however, have limitations when the interest is in localizing the virus within the tissue and identifying the specific cell types involved in viral dissemination. Chromogenic <jats:italic>in situ<\/jats:italic> hybridization (CISH) and immunohistochemistry (IHC) are common histological techniques used for intracellular localization of RNA and protein expression, respectively. The combined use of CISH and IHC is important to obtain information about RNA replication and the location of infected target cells involved in Zika virus neuropathogenesis. There are no reports, however, of detailed procedures for the simultaneous detection of Zika virus RNA and proteins in formalin\u2010fixed paraffin\u2010embedded (FFPE) samples. Furthermore, the chromogenic detection methods for Zika virus RNA published thus far use expensive commercial kits, limiting their widespread use. As an alternative, we describe here a detailed and cost\u2010effective step\u2010by\u2010step procedure for the simultaneous detection of Zika virus RNA and proteins in FFPE samples. First, we describe how to synthesize and purify homemade RNA probes conjugated with digoxygenin. Then, we outline the steps to perform the chromogenic detection of Zika virus RNA using these probes, and how to combine this technique with the immunodetection of viral antigens. To illustrate the entire workflow, we use FFPE samples derived from infected Vero cells as well as from human and mouse brain tissues. These methods are highly adaptable and can be used to study Zika virus or even other viruses of public health relevance, providing an optimal and economical alternative for laboratories with limited resources. \u00a9 2021 Wiley Periodicals LLC.<\/jats:p><jats:p><jats:bold>Basic Protocol 1<\/jats:bold>: Synthesis of RNA probes conjugated with digoxigenin (DIG)<\/jats:p><jats:p><jats:bold>Basic Protocol 2<\/jats:bold>: Simultaneous detection of ZIKV RNA and proteins in FFPE cell blocks and tissues<\/jats:p>","DOI":"10.1002\/cpz1.319","type":"journal-article","created":{"date-parts":[[2021,12,22]],"date-time":"2021-12-22T17:15:34Z","timestamp":1640193334000},"source":"Crossref","is-referenced-by-count":2,"title":["CISH and IHC for the Simultaneous Detection of ZIKV RNA and Antigens in Formalin\u2010Fixed Paraffin\u2010Embedded Cell Blocks and Tissues"],"prefix":"10.1002","volume":"1","author":[{"given":"Sheryll","family":"Corchuelo","sequence":"first","affiliation":[{"name":"Grupo de Morfolog\u00eda Celular, Direcci\u00f3n de Investigaci\u00f3n en Salud P\u00fablica Instituto Nacional de Salud  Bogot\u00e1 DC Colombia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Claudia Y.","family":"G\u00f3mez","sequence":"additional","affiliation":[{"name":"Grupo de Morfolog\u00eda Celular, Direcci\u00f3n de Investigaci\u00f3n en Salud P\u00fablica Instituto Nacional de Salud  Bogot\u00e1 DC Colombia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alicia A.","family":"Rosales","sequence":"additional","affiliation":[{"name":"Grupo de Morfolog\u00eda Celular, Direcci\u00f3n de Investigaci\u00f3n en Salud P\u00fablica Instituto Nacional de Salud  Bogot\u00e1 DC Colombia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gerardo","family":"Santamaria","sequence":"additional","affiliation":[{"name":"Grupo de Morfolog\u00eda Celular, Direcci\u00f3n de Investigaci\u00f3n en Salud P\u00fablica Instituto Nacional de Salud  Bogot\u00e1 DC Colombia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jorge Alonso","family":"Rivera","sequence":"additional","affiliation":[{"name":"Grupo de Morfolog\u00eda Celular, Direcci\u00f3n de Investigaci\u00f3n en Salud P\u00fablica Instituto Nacional de Salud  Bogot\u00e1 DC Colombia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Edgar Parra","family":"Saad","sequence":"additional","affiliation":[{"name":"Grupo de Patolog\u00eda, Direcci\u00f3n de Redes en Salud P\u00fablica Instituto Nacional de Salud  Bogot\u00e1 DC Colombia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Orlando","family":"Torres\u2010Fern\u00e1ndez","sequence":"additional","affiliation":[{"name":"Grupo de Morfolog\u00eda Celular, Direcci\u00f3n de Investigaci\u00f3n en Salud P\u00fablica Instituto Nacional de Salud  Bogot\u00e1 DC Colombia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Aura Caterine","family":"Rengifo","sequence":"additional","affiliation":[{"name":"Grupo de Morfolog\u00eda Celular, Direcci\u00f3n de Investigaci\u00f3n en Salud P\u00fablica Instituto Nacional de Salud  Bogot\u00e1 DC Colombia"},{"name":"Doctorado en Ciencias Biom\u00e9dicas Universidad Nacional de Colombia  Bogot\u00e1 DC Colombia"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2021,12,22]]},"reference":[{"issue":"1","key":"e_1_2_8_2_1","first-page":"95","article-title":"Recomendaciones sobre los aspectos \u00e9ticos de las colecciones de muestras y bancos de materiales humanos con fines de investigaci\u00f3n biom\u00e9dica","volume":"1","author":"Alonso M. A.","year":"2007","journal-title":"Revista Espa\u00f1ola de Salud P\u00fablica"},{"key":"e_1_2_8_3_1","doi-asserted-by":"publisher","DOI":"10.30699\/ijp.2017.24870"},{"key":"e_1_2_8_4_1","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-61779-897-9_3"},{"key":"e_1_2_8_5_1","doi-asserted-by":"publisher","DOI":"10.1111\/bpa.12644"},{"key":"e_1_2_8_6_1","doi-asserted-by":"publisher","DOI":"10.3201\/eid2303.161499"},{"key":"e_1_2_8_7_1","doi-asserted-by":"publisher","DOI":"10.1002\/cjp2.84"},{"key":"e_1_2_8_8_1","doi-asserted-by":"publisher","DOI":"10.1016\/S1473\u20103099(16)00095\u20105"},{"key":"e_1_2_8_9_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymeth.2014.04.006"},{"key":"e_1_2_8_10_1","doi-asserted-by":"publisher","DOI":"10.1021\/la1031703"},{"key":"e_1_2_8_11_1","doi-asserted-by":"publisher","DOI":"10.1007\/s10529\u2010013\u20101249\u20108"},{"key":"e_1_2_8_12_1","doi-asserted-by":"publisher","DOI":"10.1016\/0035\u20109203(52)90042\u20104"},{"key":"e_1_2_8_13_1","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa1601824"},{"key":"e_1_2_8_14_1","doi-asserted-by":"publisher","DOI":"10.1895\/wormbook.1.105.1"},{"key":"e_1_2_8_15_1","first-page":"23","article-title":"Fundamentals of in situ hybridization: A review","volume":"1","author":"Ehtisham M.","year":"2016","journal-title":"International Research Journal of Clinical Medicine"},{"key":"e_1_2_8_16_1","doi-asserted-by":"publisher","DOI":"10.19052\/mv.2299"},{"key":"e_1_2_8_17_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.63.2.378"},{"volume-title":"Guide for the care and use of laboratory animals","year":"2011","author":"Garber J. C.","key":"e_1_2_8_18_1"},{"key":"e_1_2_8_19_1","unstructured":"Garces Giraldo L. L\u00f3pez A. Jovany P.&Aguirre S.(2018).Bio\u00e9tica en la experimentaci\u00f3n con animales: Una mirada desde las normas para Colombia Bioethics in the experimentation with animals: A look from the norms for Colombia. Available athttps:\/\/www.researchgate.net\/publication\/333264292_Bioetica_en_la_experimentacion_con_animales_una_mirada_desde_las_normas_para_Colombia_Bioethics_in_the_experimentation_with_animals_a_look_from_the_norms_for_Colombia. doi:10.13140\/RG.2.2.29515.64806."},{"key":"e_1_2_8_20_1","doi-asserted-by":"publisher","DOI":"10.1111\/j.1365\u20102559.2012.04237.x"},{"key":"e_1_2_8_21_1","doi-asserted-by":"publisher","DOI":"10.1002\/pmic.201800309"},{"key":"e_1_2_8_22_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0120120"},{"key":"e_1_2_8_23_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.virol.2014.10.037"},{"key":"e_1_2_8_24_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1007507"},{"key":"e_1_2_8_25_1","first-page":"343","article-title":"Chromogenic in situ hybridization and FISH in pathology","volume":"204","author":"Hsi B. L.","year":"2002","journal-title":"Methods in Molecular Biology"},{"key":"e_1_2_8_26_1","doi-asserted-by":"publisher","DOI":"10.3389\/fnins.2018.00266"},{"key":"e_1_2_8_27_1","doi-asserted-by":"publisher","DOI":"10.1016\/0890\u20108508(92)90021\u2010O"},{"key":"e_1_2_8_28_1","doi-asserted-by":"publisher","DOI":"10.1002\/ar.22944"},{"key":"e_1_2_8_29_1","doi-asserted-by":"publisher","DOI":"10.1086\/383043"},{"key":"e_1_2_8_30_1","doi-asserted-by":"publisher","DOI":"10.1097\/PAI.0000000000000811"},{"key":"e_1_2_8_31_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.virol.2004.03.009"},{"key":"e_1_2_8_32_1","doi-asserted-by":"publisher","DOI":"10.15585\/mmwr.mm6509e2"},{"key":"e_1_2_8_33_1","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2017.00108"},{"key":"e_1_2_8_34_1","doi-asserted-by":"publisher","DOI":"10.1093\/molbev\/msy096"},{"key":"e_1_2_8_35_1","doi-asserted-by":"publisher","DOI":"10.1128\/MRA.01719\u201018"},{"key":"e_1_2_8_36_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.humpath.2007.04.011"},{"key":"e_1_2_8_37_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.diagmicrobio.2018.06.019"},{"key":"e_1_2_8_38_1","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa1600651"},{"key":"e_1_2_8_39_1","doi-asserted-by":"publisher","DOI":"10.1128\/CMR.00072\u201015"},{"key":"e_1_2_8_40_1","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMcibr1605445"},{"key":"e_1_2_8_41_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00769\u2010003\u20100588\u2010x"},{"volume-title":"Gu\u00eda para la vigilancia de la enfermedad por el virus del Zika y sus complicaciones","year":"2018","author":"OPS","key":"e_1_2_8_42_1"},{"key":"e_1_2_8_43_1","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMra1602113"},{"key":"e_1_2_8_44_1","doi-asserted-by":"publisher","DOI":"10.3390\/v10070384"},{"key":"e_1_2_8_45_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0007468"},{"key":"e_1_2_8_46_1","doi-asserted-by":"publisher","DOI":"10.1007\/s13337\u2010012\u20100105\u20100"},{"key":"e_1_2_8_47_1","doi-asserted-by":"publisher","DOI":"10.1097\/00019606\u2010200303000\u201000001"},{"key":"e_1_2_8_48_1","doi-asserted-by":"publisher","DOI":"10.1590\/1414\u2010431X20132483"},{"key":"e_1_2_8_49_1","doi-asserted-by":"publisher","DOI":"10.1007\/978\u20101\u20104939\u20108935\u20105_22"},{"key":"e_1_2_8_50_1","doi-asserted-by":"publisher","DOI":"10.15585\/mmwr.mm6503e2"},{"key":"e_1_2_8_51_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0218539"},{"key":"e_1_2_8_52_1","first-page":"235","article-title":"The use of combined immunohistochemical labeling and in situ hybridization to colocalize mRNA and protein in tissue sections","volume":"326","author":"Smith M. D.","year":"2006","journal-title":"Methods in Molecular Biology"},{"key":"e_1_2_8_53_1","doi-asserted-by":"publisher","DOI":"10.1007\/978\u20103\u2010642\u201069159\u20101_1"},{"key":"e_1_2_8_54_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0002\u20109440(10)64785\u20102"},{"key":"e_1_2_8_55_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.celrep.2018.03.080"},{"key":"e_1_2_8_56_1","doi-asserted-by":"publisher","DOI":"10.1101\/785220"},{"key":"e_1_2_8_57_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ajog.2018.06.005"},{"key":"e_1_2_8_58_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0140\u20106736(16)00273\u20107"},{"key":"e_1_2_8_59_1","first-page":"2.5A.1","article-title":"Agarose gel electrophoresis","volume":"51","author":"Voytas D.","year":"2001","journal-title":"Current Protocols in Molecular Biology"},{"key":"e_1_2_8_60_1","doi-asserted-by":"publisher","DOI":"10.1038\/cr.2016.58"},{"key":"e_1_2_8_61_1","doi-asserted-by":"publisher","DOI":"10.1002\/0471142727.mb0101s07"},{"key":"e_1_2_8_62_1","doi-asserted-by":"publisher","DOI":"10.1007\/s13365\u2010017\u20100514\u20103"}],"container-title":["Current Protocols"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cpz1.319","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/full-xml\/10.1002\/cpz1.319","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cpz1.319","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,8,22]],"date-time":"2023-08-22T03:16:02Z","timestamp":1692674162000},"score":1,"resource":{"primary":{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/10.1002\/cpz1.319"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,12]]},"references-count":61,"journal-issue":{"issue":"12","published-print":{"date-parts":[[2021,12]]}},"alternative-id":["10.1002\/cpz1.319"],"URL":"https:\/\/doi.org\/10.1002\/cpz1.319","archive":["Portico"],"relation":{},"ISSN":["2691-1299","2691-1299"],"issn-type":[{"type":"print","value":"2691-1299"},{"type":"electronic","value":"2691-1299"}],"subject":[],"published":{"date-parts":[[2021,12]]},"article-number":"e319"}}