{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,21]],"date-time":"2025-10-21T14:51:46Z","timestamp":1761058306183,"version":"build-2065373602"},"reference-count":12,"publisher":"Wiley","issue":"1","license":[{"start":{"date-parts":[[2005,7,15]],"date-time":"2005-07-15T00:00:00Z","timestamp":1121385600000},"content-version":"vor","delay-in-days":75,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Microscopy Res &amp;amp; Technique"],"published-print":{"date-parts":[[2005,5]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Immune\u2010cell infiltration is frequently seen within human solid tumors. A detailed phenotypic analysis of these cells may aid in the understanding of an antitumor immune response. Standard hematoxylin\/eosin and conventional immunohistochemical stainings are helpful, but have major limitations in the number of markers that can be identified and localized per tissue section. Therefore, we developed a combined fluorescence and brightfield microscopic technique by using both immunofluorescence and immunogold\u2010silver methods, thereby discriminating three different leukocyte markers plus one tumor marker simultaneously in a single section. This enabled us to study both phenotype and location of infiltrating immune cells in colorectal tumors. We used a two\u2010step staining in which primary and secondary antibodies were selected for minimal cross\u2010reactivity. Furthermore, the secondary fluorescent antibody conjugates were selected for minimal spectral overlap. For computer\u2010assisted analysis the brightfield microscopy image was combined with the fluorescence microscopy images. This combination of techniques provides a powerful tool for detailed multiparameter microscopic analysis of formalin\u2010fixed, paraffin\u2010embedded tissue sections in general and for tumor\u2010immune cell infiltration in particular. Microsc. Res. Tech. 67:15\u201321, 2005. \u00a9 2005 Wiley\u2010Liss, Inc.<\/jats:p>","DOI":"10.1002\/jemt.20181","type":"journal-article","created":{"date-parts":[[2005,7,15]],"date-time":"2005-07-15T15:18:03Z","timestamp":1121440683000},"page":"15-21","source":"Crossref","is-referenced-by-count":10,"title":["Four\u2010color staining combining fluorescence and brightfield microscopy for simultaneous immune cell phenotyping and localization in tumor tissue sections"],"prefix":"10.1002","volume":"67","author":[{"given":"Ronald L.P.","family":"van Vlierberghe","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Maro H.","family":"Sandel","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Frans A.","family":"Prins","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Liselot B.J.","family":"van Iersel","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Cornelis J.H.","family":"van de Velde","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rob A.E.M.","family":"Tollenaar","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Peter J.K.","family":"Kuppen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2005,7,15]]},"reference":[{"key":"e_1_2_1_2_1","first-page":"239","article-title":"Differences in the germinal centres of palatine tonsils and lymph nodes","volume":"43","author":"Brachtel EF","year":"1996","journal-title":"Scand J Immunol"},{"key":"e_1_2_1_3_1","doi-asserted-by":"publisher","DOI":"10.1097\/00000478-198612000-00001"},{"key":"e_1_2_1_4_1","doi-asserted-by":"publisher","DOI":"10.1046\/j.1365-3083.2001.00838.x"},{"key":"e_1_2_1_5_1","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2172-6-3"},{"key":"e_1_2_1_6_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00262-002-0363-6"},{"key":"e_1_2_1_7_1","doi-asserted-by":"publisher","DOI":"10.1038\/labinvest.3700055"},{"key":"e_1_2_1_8_1","first-page":"3491","article-title":"CD8+ T cells infiltrated within cancer cell nests as a prognostic factor in human colorectal cancer","volume":"58","author":"Naito Y","year":"1998","journal-title":"Cancer Res"},{"key":"e_1_2_1_9_1","first-page":"120","article-title":"Multiple fluorescence and reflectance simultaneous detection by confocal microscopy of HaeIII digested DNA sequences","volume":"71","author":"Neri LM","year":"1996","journal-title":"Eur J Cell Biol"},{"key":"e_1_2_1_10_1","doi-asserted-by":"publisher","DOI":"10.1038\/ki.1996.37"},{"key":"e_1_2_1_11_1","doi-asserted-by":"publisher","DOI":"10.1177\/43.1.7822758"},{"key":"e_1_2_1_12_1","doi-asserted-by":"publisher","DOI":"10.1016\/0022-1759(89)90117-8"},{"key":"e_1_2_1_13_1","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.10026"}],"container-title":["Microscopy Research and Technique"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/api.wiley.com\/onlinelibrary\/tdm\/v1\/articles\/10.1002%2Fjemt.20181","content-type":"unspecified","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/analyticalsciencejournals.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/jemt.20181","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,16]],"date-time":"2025-10-16T19:09:50Z","timestamp":1760641790000},"score":1,"resource":{"primary":{"URL":"https:\/\/analyticalsciencejournals.onlinelibrary.wiley.com\/doi\/10.1002\/jemt.20181"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2005,5]]},"references-count":12,"journal-issue":{"issue":"1","published-print":{"date-parts":[[2005,5]]}},"alternative-id":["10.1002\/jemt.20181"],"URL":"https:\/\/doi.org\/10.1002\/jemt.20181","archive":["Portico"],"relation":{},"ISSN":["1059-910X","1097-0029"],"issn-type":[{"type":"print","value":"1059-910X"},{"type":"electronic","value":"1097-0029"}],"subject":[],"published":{"date-parts":[[2005,5]]}}}