{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,13]],"date-time":"2026-09-13T11:05:14Z","timestamp":1789297514946,"version":"build-2803163510"},"reference-count":48,"publisher":"SAGE Publications","issue":"5","license":[{"start":{"date-parts":[[2026,3,21]],"date-time":"2026-03-21T00:00:00Z","timestamp":1774051200000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.sagepub.com\/page\/policies\/text-and-data-mining-license"}],"funder":[{"name":"Center for Integrative Mammalian Research"},{"name":"Center for One-Health, Zoonosis, and Tropical Medicine"},{"DOI":"10.13039\/100021039","name":"school of veterinary medicine, ross university","doi-asserted-by":"publisher","award":["41008-2022"],"award-info":[{"award-number":["41008-2022"]}],"id":[{"id":"10.13039\/100021039","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.sagepub.com"],"crossmark-restriction":true},"short-container-title":["J VET Diagn Invest"],"published-print":{"date-parts":[[2026,9]]},"abstract":"<jats:p>Fluorescence in situ hybridization (FISH) can be used to detect intracellular bacteria in formalin-fixed paraffin-embedded (FFPE) tissues. However, this technique has rarely been applied to samples of feline heart, liver, or kidney to investigate the relationship between intracellular bacteria and chronic inflammation within these organs. Our objective was to develop a robust FISH protocol to detect intracellular bacteria within medium-density FFPE feline tissue samples. Cy3-labeled eubacterial and non-eubacterial probes were applied to samples of monkey intestine, feline intestine, and feline liver, in which bacteria had been visualized on routine histopathology. Although initial tests failed, the addition of pepsin pre-digestion to the test protocol and the adjustment of stringency ensured consistent results. All positive results were confirmed using Gram staining and non-eubacterial probes applied to sequential samples of the 3 tissues. Our FISH protocol using Cy3-labeled eubacterial probes reliably and consistently detected bacteria within FFPE samples of the 3 tissues. The addition of 20-min pepsin pre-digestion and the adjustment of the formamide concentration within the hybridization buffer to 40% were pivotal to the successful use of the protocol. Species-specific probes, along with PCR, culture, and special staining, could be considered to increase sensitivity and specificity of FISH when used for the detection of organisms within tissues.<\/jats:p>","DOI":"10.1177\/10406387261432648","type":"journal-article","created":{"date-parts":[[2026,3,21]],"date-time":"2026-03-21T09:31:27Z","timestamp":1774085487000},"page":"873-884","update-policy":"https:\/\/doi.org\/10.1177\/sage-journals-update-policy","source":"Crossref","is-referenced-by-count":0,"title":["Development of a fluorescence in situ hybridization protocol for use with formalin-fixed paraffin-embedded samples of feline heart, liver, and kidney"],"prefix":"10.1177","volume":"38","author":[{"ORCID":"https:\/\/orcid.org\/0009-0006-0539-2690","authenticated-orcid":false,"given":"Matthew J.","family":"Rolph","sequence":"first","affiliation":[{"name":"Departments of Clinical Sciences, Ross University School of Veterinary Medicine, Basseterre, St Kitts, West Indies"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2903-1535","authenticated-orcid":false,"given":"Pompei","family":"Bolfa","sequence":"additional","affiliation":[{"name":"Biomedical Sciences, Ross University School of Veterinary Medicine, Basseterre, St Kitts, West Indies"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sarah M.","family":"Cavanaugh","sequence":"additional","affiliation":[{"name":"Departments of Clinical Sciences, Ross University School of Veterinary Medicine, Basseterre, St Kitts, West Indies"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David","family":"Hilchie","sequence":"additional","affiliation":[{"name":"Biomedical Sciences, Ross University School of Veterinary Medicine, Basseterre, St Kitts, West Indies"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4660-5676","authenticated-orcid":false,"given":"Kerry E.","family":"Rolph","sequence":"additional","affiliation":[{"name":"Departments of Clinical Sciences, Ross University School of Veterinary Medicine, Basseterre, St Kitts, West Indies"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"179","published-online":{"date-parts":[[2026,3,21]]},"reference":[{"key":"e_1_3_3_2_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0230057"},{"key":"e_1_3_3_3_2","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-0716-1115-9_1"},{"key":"e_1_3_3_4_2","doi-asserted-by":"publisher","DOI":"10.1093\/ajcp\/aqx094"},{"key":"e_1_3_3_5_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-019-55049-2"},{"key":"e_1_3_3_6_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0168-6496(02)00461-0"},{"key":"e_1_3_3_7_2","doi-asserted-by":"publisher","DOI":"10.3389\/fmed.2017.00087"},{"key":"e_1_3_3_8_2","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-0716-0623-0_4"},{"key":"e_1_3_3_9_2","doi-asserted-by":"crossref","unstructured":"Bridger JM Volpi EV eds. Fluorescence In Situ Hybridization (FISH): Protocols and Applications. (Vol. 659 Methods in Molecular Biology). Humana Press 2010.","DOI":"10.1007\/978-1-60761-789-1"},{"key":"e_1_3_3_10_2","unstructured":"Cambio Ltd. Fluorescence in situ hybridisation protocol for whole chromosome paints November 2009. https:\/\/www.cambio.co.uk\/help\/protocols\/WCP-Hybridisation.pdf"},{"key":"e_1_3_3_11_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.toxrep.2022.04.012"},{"key":"e_1_3_3_12_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkx471"},{"key":"e_1_3_3_13_2","doi-asserted-by":"crossref","unstructured":"Farrell RE. RNA Methodologies: A Laboratory Guide for Isolation and Characterization. 4th ed. Elsevier 2010:173\u2013178.","DOI":"10.1016\/B978-0-12-374727-3.00008-5"},{"key":"e_1_3_3_14_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.280.5363.585"},{"key":"e_1_3_3_15_2","first-page":"6564","article-title":"Fluorescence in situ hybridization on DNA halo preparations to reveal whole chromosomes, telomeres and gene loci. J Vis Exp 2021;169:62017","volume":"196","author":"Godwin LS","year":"2023","unstructured":"Godwin LS, et al. Fluorescence in situ hybridization on DNA halo preparations to reveal whole chromosomes, telomeres and gene loci. J Vis Exp 2021;169:62017. Erratum in: J Vis Exp 2023;196:6564.","journal-title":"J Vis Exp"},{"key":"e_1_3_3_16_2","first-page":"17","article-title":"FISH variants. Fluorescence in-situ hybridization (FISH) for microbial cells","volume":"2246","author":"Guimar\u00e3es NM","year":"2021","unstructured":"Guimar\u00e3es NM, et al. FISH variants. Fluorescence in-situ hybridization (FISH) for microbial cells. Methods Mol Biol 2021;2246:17\u201333.","journal-title":"Methods Mol Biol"},{"key":"e_1_3_3_17_2","doi-asserted-by":"publisher","DOI":"10.1186\/1749-8090-7-121"},{"key":"e_1_3_3_18_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/4.10.3537"},{"key":"e_1_3_3_19_2","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-0716-0623-0_1"},{"key":"e_1_3_3_20_2","doi-asserted-by":"publisher","DOI":"10.5483\/BMBRep.2013.46.2.016"},{"key":"e_1_3_3_21_2","doi-asserted-by":"publisher","DOI":"10.1007\/BF00291428"},{"key":"e_1_3_3_22_2","doi-asserted-by":"publisher","DOI":"10.1002\/jemt.24702"},{"key":"e_1_3_3_23_2","doi-asserted-by":"publisher","DOI":"10.1242\/jcs.00633"},{"key":"e_1_3_3_24_2","doi-asserted-by":"crossref","unstructured":"Liehr T ed. Fluorescence In Situ Hybridization (FISH): Application Guide. 2nd ed. (Springer Protocols). Springer 2017.","DOI":"10.1007\/978-3-662-52959-1"},{"key":"e_1_3_3_25_2","first-page":"109","volume-title":"Fluorescence In Situ Hybridization (FISH)","author":"Liehr T","unstructured":"Liehr T, et al. The standard FISH procedure. In: Liehr T ed. Fluorescence In Situ Hybridization (FISH). 2nd ed. Springer, 2017:109\u2013118.","edition":"2"},{"key":"e_1_3_3_26_2","doi-asserted-by":"publisher","DOI":"10.1016\/bs.mie.2016.03.020"},{"key":"e_1_3_3_27_2","doi-asserted-by":"publisher","DOI":"10.1177\/1040638711425584"},{"key":"e_1_3_3_28_2","first-page":"1","article-title":"Detection of tissue-resident bacteria in bladder biopsies by 16s rRNA fluorescence in situ hybridization","volume":"152","author":"Neugent ML","year":"2019","unstructured":"Neugent ML, et al. Detection of tissue-resident bacteria in bladder biopsies by 16s rRNA fluorescence in situ hybridization. J Vis Exp 2019;152:1\u20136.","journal-title":"J Vis Exp"},{"key":"e_1_3_3_29_2","unstructured":"Nielsen BS Jones J eds. In Situ Hybridization Protocols. 5th ed. Vol. 2148. Springer 2020:7\u20139 74 80 88 91 92 328."},{"key":"e_1_3_3_30_2","doi-asserted-by":"publisher","DOI":"10.1093\/femsre\/fuy040"},{"key":"e_1_3_3_31_2","doi-asserted-by":"publisher","DOI":"10.3390\/vetsci11010052"},{"key":"e_1_3_3_32_2","doi-asserted-by":"publisher","DOI":"10.1177\/1098612X251396969"},{"key":"e_1_3_3_33_2","doi-asserted-by":"publisher","DOI":"10.5858\/arpa.2016-0202-RA"},{"key":"e_1_3_3_34_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0075120"},{"key":"e_1_3_3_35_2","unstructured":"Sigma-Aldrich. User guide - eubacteria fluorescent in situ hybridization (FISH) probe for bacterial detection. Merk KGaA 2021:1\u20138. https:\/\/drive.google.com\/file\/d\/1WRGAFN7k90Tr7q6xVYRxALem-zWTcmV4\/view?usp=drive_link"},{"key":"e_1_3_3_36_2","doi-asserted-by":"publisher","DOI":"10.1002\/jnr.22191"},{"key":"e_1_3_3_37_2","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.00067-06"},{"key":"e_1_3_3_38_2","first-page":"537","volume-title":"Theory and Practice of Histological Techniques","author":"Sterchi DL","unstructured":"Sterchi DL. Molecular pathology\u2014in situ hybridization. In: Bancroft JD, Gamble M, eds. Theory and Practice of Histological Techniques. 6th ed. Churchill Livingston, 2008:537\u2013588.","edition":"6"},{"key":"e_1_3_3_39_2","doi-asserted-by":"publisher","DOI":"10.1007\/s00428-015-1777-9"},{"key":"e_1_3_3_40_2","doi-asserted-by":"publisher","DOI":"10.1177\/1098612X13498249"},{"key":"e_1_3_3_41_2","first-page":"71","volume-title":"Molecular Diagnostics: Techniques and Applications for the Clinical Laboratory","author":"Unger ER","unstructured":"Unger ER, et al. In situ hybridization: principles and applications. In: Grody WW, et al., eds. Molecular Diagnostics: Techniques and Applications for the Clinical Laboratory: Academic Press, 2010:71\u201379."},{"key":"e_1_3_3_42_2","doi-asserted-by":"publisher","DOI":"10.3390\/molecules26133874"},{"key":"e_1_3_3_43_2","doi-asserted-by":"publisher","DOI":"10.2144\/000112811"},{"key":"e_1_3_3_44_2","doi-asserted-by":"publisher","DOI":"10.2353\/jmoldx.2007.060128"},{"key":"e_1_3_3_45_2","doi-asserted-by":"publisher","DOI":"10.1038\/s42003-018-0218-0"},{"key":"e_1_3_3_46_2","doi-asserted-by":"publisher","DOI":"10.1177\/0300060520931259"},{"key":"e_1_3_3_47_2","doi-asserted-by":"publisher","DOI":"10.7717\/peerj.8806"},{"key":"e_1_3_3_48_2","article-title":"Robustness of RNA sequencing on older formalin-fixed paraffin-embedded tissue from high-grade ovarian serous adenocarcinomas","volume":"14","author":"Zhao Y","year":"2019","unstructured":"Zhao Y, et al. Robustness of RNA sequencing on older formalin-fixed paraffin-embedded tissue from high-grade ovarian serous adenocarcinomas. PLoS One 2019;14:e0216050.","journal-title":"PLoS One"},{"key":"e_1_3_3_49_2","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-61779-074-4_14"}],"container-title":["Journal of Veterinary Diagnostic Investigation"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/journals.sagepub.com\/doi\/pdf\/10.1177\/10406387261432648","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/journals.sagepub.com\/doi\/full-xml\/10.1177\/10406387261432648","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/journals.sagepub.com\/doi\/pdf\/10.1177\/10406387261432648","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,13]],"date-time":"2026-09-13T10:19:26Z","timestamp":1789294766000},"score":1,"resource":{"primary":{"URL":"https:\/\/journals.sagepub.com\/doi\/10.1177\/10406387261432648"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,3,21]]},"references-count":48,"journal-issue":{"issue":"5","published-print":{"date-parts":[[2026,9]]}},"alternative-id":["10.1177\/10406387261432648"],"URL":"https:\/\/doi.org\/10.1177\/10406387261432648","relation":{},"ISSN":["1040-6387","1943-4936"],"issn-type":[{"value":"1040-6387","type":"print"},{"value":"1943-4936","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,3,21]]}}}