{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,16]],"date-time":"2026-01-16T17:51:37Z","timestamp":1768585897585,"version":"3.49.0"},"reference-count":47,"publisher":"Wiley","issue":"1","license":[{"start":{"date-parts":[[2024,6,10]],"date-time":"2024-06-10T00:00:00Z","timestamp":1717977600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004284","name":"R.T. Hall Trust","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100004284","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Journal of Microscopy"],"published-print":{"date-parts":[[2024,10]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Immunohistochemistry (IHC) and immunofluorescence (IF) are crucial techniques for studying cardiac physiology and disease. The accuracy of these techniques is dependent on various aspects of sample preparation and processing. However, standardised protocols for sample preparation of tissues, particularly for fresh\u2010frozen human left ventricle (LV) tissue, have yet to be established and could potentially lead to differences in staining and interpretation. Thus, this study aimed to optimise the reproducibility and quality of IF staining in fresh\u2010frozen human LV tissue by systematically investigating crucial aspects of the sample preparation process. To achieve this, we subjected fresh\u2010frozen human LV tissue to different fixation protocols, primary antibody incubation temperatures, antibody penetration reagents, and fluorescent probes. We found that neutral buffered formalin fixation reduced image artefacts and improved antibody specificity compared to both methanol and acetone fixation. Additionally, incubating primary antibodies at 37\u00b0C for 3 h improved fluorescence intensity compared to the commonly practised 4\u00b0C overnight incubation. Furthermore, we found that DeepLabel, an antibody penetration reagent, and smaller probes, such as fragmented antibodies and Affimers, improved the visualisation depth of cardiac structures. DeepLabel also improved antibody penetration in CUBIC cleared thick LV tissue fragments. Thus, our data underscores the importance of standardised protocols in IF staining and provides various means of improving staining quality. In addition to contributing to cardiac research by providing methodologies for IF, the findings and processes presented herein also establish a framework by which staining of other tissues may be optimised.<\/jats:p>","DOI":"10.1111\/jmi.13336","type":"journal-article","created":{"date-parts":[[2024,6,10]],"date-time":"2024-06-10T15:22:01Z","timestamp":1718032921000},"page":"34-47","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["A comparison of fixation and immunofluorescence protocols for successful reproducibility and improved signal in human left ventricle cardiac tissue"],"prefix":"10.1111","volume":"296","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3148-1615","authenticated-orcid":false,"given":"Matthew","family":"Taper","sequence":"first","affiliation":[{"name":"Faculty of Medicine and Health School of Medical Sciences, The University of Sydney  Sydney Australia"},{"name":"Centre for Heart Failure and 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