{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,12]],"date-time":"2025-10-12T02:21:51Z","timestamp":1760235711779,"version":"build-2065373602"},"reference-count":13,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2021,9,13]],"date-time":"2021-09-13T00:00:00Z","timestamp":1631491200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Background: There is increasing evidence that identification of SARS-CoV-2 virions by transmission electron microscopy could be misleading due to the similar morphology of virions and ubiquitous cell structures. This study thus aimed to establish methods for indisputable proof of the presence of SARS-CoV-2 virions in the observed tissue. Methods: We developed a variant of the correlative microscopy approach for SARS-CoV-2 protein identification using immunohistochemical labelling of SARS-CoV-2 proteins on light and electron microscopy levels. We also performed immunogold labelling of SARS-CoV-2 virions. Results: Immunohistochemistry (IHC) of SARS-CoV-2 nucleocapsid proteins and subsequent correlative microscopy undoubtedly proved the presence of SARS-CoV-2 virions in the analysed human nasopharyngeal tissue. The presence of SARS-CoV-2 virions was also confirmed by immunogold labelling for the first time. Conclusions: Immunoelectron microscopy is the most reliable method for distinguishing intracellular viral particles from normal cell structures of similar morphology and size as virions. Furthermore, we developed a variant of correlative microscopy that allows pathologists to check the results of IHC performed first on routinely used paraffin-embedded samples, followed by semithin, and finally by ultrathin sections. Both methodological approaches indisputably proved the presence of SARS-CoV-2 virions in cells.<\/jats:p>","DOI":"10.3390\/v13091816","type":"journal-article","created":{"date-parts":[[2021,9,14]],"date-time":"2021-09-14T03:46:14Z","timestamp":1631591174000},"page":"1816","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Just Seeing Is Not Enough for Believing: Immunolabelling as Indisputable Proof of SARS-CoV-2 Virions in Infected Tissue"],"prefix":"10.3390","volume":"13","author":[{"given":"Andreja","family":"Erman","sequence":"first","affiliation":[{"name":"Institute of Cell Biology, Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Karmen","family":"Wechtersbach","sequence":"additional","affiliation":[{"name":"Institute of Pathology, Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Daniel","family":"Velkavrh","sequence":"additional","affiliation":[{"name":"Institute of Pathology, Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jerica","family":"Ple\u0161ko","sequence":"additional","affiliation":[{"name":"Institute of Pathology, Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Maja","family":"Frelih","sequence":"additional","affiliation":[{"name":"Institute of Pathology, Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1893-4349","authenticated-orcid":false,"given":"Nika","family":"Kojc","sequence":"additional","affiliation":[{"name":"Institute of Pathology, Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2021,9,13]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"219","DOI":"10.1016\/j.kint.2020.04.003","article-title":"Renal Histopathological Analysis of 26 Postmortem Findings of Patients with COVID-19 in China","volume":"98","author":"Su","year":"2020","journal-title":"Kidney Int."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"e311","DOI":"10.1111\/jdv.17123","article-title":"COVID-19 and HHV8 first spotted together: An affair under electron microscopy","volume":"35","author":"Leoni","year":"2021","journal-title":"J. Eur. Acad. Dermatol. Venereol."},{"key":"ref_3","first-page":"5186","article-title":"SARS-CoV-2 identification in lungs, heart and kidney specimens by transmission and scanning electron microscopy","volume":"24","author":"Pesaresi","year":"2020","journal-title":"Eur. Rev. Med. Pharmacol. Sci."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"231","DOI":"10.1016\/j.kint.2020.05.004","article-title":"Visualization of putative coronavirus in kidney","volume":"98","author":"Miller","year":"2020","journal-title":"Kidney Int."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"506","DOI":"10.1016\/j.kint.2020.05.021","article-title":"Am I a coronavirus?","volume":"98","author":"Smith","year":"2020","journal-title":"Kidney Int."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1608","DOI":"10.1016\/j.ekir.2020.07.003","article-title":"SARS-CoV-2 Virions or Ubiquitous Cell Structures? Actual Dilemma in COVID-19 Era","volume":"5","author":"Frelih","year":"2020","journal-title":"Kidney Int. Rep."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"552","DOI":"10.1128\/CMR.00027-09","article-title":"Modern Uses of Electron Microscopy for Detection of Viruses","volume":"22","author":"Goldsmith","year":"2009","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1083\/jcb.148.1.45","article-title":"Correlative Light-Electron Microscopy Reveals the Tubular-Saccular Ultrastructure of Carriers Operating between Golgi Apparatus and Plasma Membrane","volume":"148","author":"Polishchuk","year":"2000","journal-title":"J. Cell Biol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1046\/j.1365-2818.2000.00704.x","article-title":"Immunocytochemical strategies for electron microscopy: Choice or compromise","volume":"199","author":"Skepper","year":"2000","journal-title":"J. Microsc."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"88","DOI":"10.1177\/0300985813503571","article-title":"Rodent Immunohistochemistry: Pitfalls and Troubleshooting","volume":"51","author":"Ward","year":"2014","journal-title":"Vet. Pathol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"115","DOI":"10.1177\/43.2.7529784","article-title":"Antigen unmasking for immunoelectron microscopy: Labeling is improved by treating with sodium ethoxide or sodium metaperiodate, then heating on retrieval medium","volume":"43","author":"Stirling","year":"1995","journal-title":"J. Histochem. Cytochem."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"347","DOI":"10.1007\/s00418-014-1263-5","article-title":"Antibodies for immunolabelling by light and electron microscopy: Not for the faint hearted","volume":"142","author":"Griffiths","year":"2014","journal-title":"Histochem. Cell Biol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"320","DOI":"10.3201\/eid1002.030913","article-title":"Ultrastructural Characterization of SARS Coronavirus","volume":"10","author":"Goldsmith","year":"2004","journal-title":"Emerg. Infect. Dis."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/13\/9\/1816\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T07:01:52Z","timestamp":1760166112000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/13\/9\/1816"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,9,13]]},"references-count":13,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2021,9]]}},"alternative-id":["v13091816"],"URL":"https:\/\/doi.org\/10.3390\/v13091816","relation":{},"ISSN":["1999-4915"],"issn-type":[{"type":"electronic","value":"1999-4915"}],"subject":[],"published":{"date-parts":[[2021,9,13]]}}}