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. 2020 Jul 14;10:11551. doi: 10.1038/s41598-020-68200-1

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Multiple cryoinjuries enhance deposition of ColXII and connective tissue in the remaining fibrotic tissue. (A) Cross sections of adult zebrafish hearts immunostained for the type XII collagen (ColXII; green) and Fibronectin (red) in uninjured hearts and at 60 dpci, following one, two, three or six cryoinjuries. The cardiac muscle is detected by F-actin staining (Phalloidin, blue). In uninjured hearts, ColXII is detected in the epicardium (E) and the junctional region (J) between the compact myocardium (CoM) and the trabecular myocardium (TrM). After cryoinjuries, the fibrotic tissue of partially regenerated hearts contains ColXII and Fibronectin (yellow through an overlay of green and red staining). (B) Cross sections of adult zebrafish hearts stained with fluorescein-conjugated lectin Ricinus communis agglutinin 1 (RCA1, green). The cardiac muscle is detected by F-actin staining (Phalloidin, red). In uninjured hearts, RCA1 labels the valve (V), the epicardium (E) and the junctional region (J) between the compact (CoM) and the trabecular myocardium (TrM). After cryoinjuries, the fibrotic tissue of partially regenerated hearts displays abundant RCA1 labelling. (C) Analysis of the percentage of ColXII-positive area per heart section. N ≥ 5. *P < 0.05; **P < 0.01; ***P < 0.001. Scatter plot of the data with a large bar indicating the mean and smaller bars representing the SEM. (D) Analysis of the percentage RCA1-positive area per heart section. N ≥ 5. *P < 0.05; **P < 0.01. Scatter plot of the data with a large bar indicating the mean and smaller bars representing the SEM.