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. 2020 Feb 10;10:2191. doi: 10.1038/s41598-020-59106-z

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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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Enhanced accumulation of autophagosomes and phagolysosomes is observed in the injured cardiac tissue upon amputation. The transgenic line Tg(b-actin2:mRFP-GFP-Lc3) allows the observation of autophagosome formation and processing in the regenerating zebrafish heart ventricle at different time points after apex amputation. Autophagosomes are depicted both as GFP- and RFP-puncta, while the lower pH-sensitivity of RFP additionally allows the visualization of phagolysosomes. Images were taken immediately after heart excision, and upon overnight incubation of the zebrafish in 2 mM CQ, which acts as a lysosomal inhibitor. Quantitative analysis revealed a significant increase in the number of both vesicles, as well as in the average autophagosome/phagolysosome size (red puncta) in the regenerating tissue, which gradually decreased over time. A schematic view of the ventricle orientation and the region of interest considered for image acquisition is shown, while higher magnifications exemplifying the accumulation of bigger autophagic vesicles are presented next to the mosaic images. Scale bars represent 200 μm. *p ≤ 0.05. N ≥ 3.