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. 2018 Sep 18;8:14017. doi: 10.1038/s41598-018-32336-y

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© The Author(s) 2018

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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High-content screening of 1351 bioactive small molecules for ROS renewal. (a) Work-flow for high-content, image-based screen. Tg(Xla.rho:EGFP); Tg(hsp70:HA-mCherry^TM); alb^−/− fish were heat shocked at 6 dpf to induce expression of HA-mCherry^TM followed by rearing in 20 μM compound until 10 dpf when tissues were fixed and processed for immunolabeling and confocal microscopy. (b) Confocal z-stack images were acquired of the GFP (green) and HA-mCherry^TM stripe (red)-expressing rods labeled with anti-Rhodopsin antibody (blue) in two regions of interest: the photoreceptor layer and ciliary margin (white-hatched boxes). (c) ROS renewal was measured within the three-dimensional z-stacks with the growth distance (D^G) representing the distance from the base of the ROS to the mCherry^TM stripe and the shedding distance (D^S) representing the distance from the mCherry^TM stripe to the tip of the outer segment. Scale bar is 10 μm. (d,e) Qualitative changes in the photoreceptor layer were documented, including the (d) accumulation of phagosomes and (e) Rhodopsin mislocalization. (f) Qualitative changes in the ciliary margin were documented, such as the size of the proliferative zone and addition of new rods during compound treatment, which can be identified by lack of the mCherry^TM stripe.