A comparison of fixation and immunofluorescence protocols for successful reproducibility and improved signal in human left ventricle cardiac tissue.

Autor: Taper M; Faculty of Medicine and Health, School of Medical Sciences, The University of Sydney, Sydney, Australia.; Centre for Heart Failure and Diseases of the Aorta, The Baird Institute, Sydney, Australia., Carrington G; Faculty of Biological Sciences, Astbury Centre for Structural Biology and the School of Molecular and Cellular Biology, University of Leeds, Leeds, UK., Peckham M; Faculty of Biological Sciences, Astbury Centre for Structural Biology and the School of Molecular and Cellular Biology, University of Leeds, Leeds, UK., Lal S; Faculty of Medicine and Health, School of Medical Sciences, The University of Sydney, Sydney, Australia.; Centre for Heart Failure and Diseases of the Aorta, The Baird Institute, Sydney, Australia.; Department of Cardiology, Royal Prince Alfred Hospital, Sydney, Australia., Hume RD; Faculty of Medicine and Health, School of Medical Sciences, The University of Sydney, Sydney, Australia.; Centre for Heart Failure and Diseases of the Aorta, The Baird Institute, Sydney, Australia.
Jazyk: angličtina
Zdroj: Journal of microscopy [J Microsc] 2024 Oct; Vol. 296 (1), pp. 34-47. Date of Electronic Publication: 2024 Jun 10.
DOI: 10.1111/jmi.13336
Abstrakt: 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-frozen 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-frozen human LV tissue by systematically investigating crucial aspects of the sample preparation process. To achieve this, we subjected fresh-frozen 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°C for 3 h improved fluorescence intensity compared to the commonly practised 4°C 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.
(© 2024 The Author(s). Journal of Microscopy published by John Wiley & Sons Ltd on behalf of Royal Microscopical Society.)
Databáze: MEDLINE
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