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Int. J. Mol. Sci. 2020, 21, 2370 16 of 20 was removed by washing twice with hybridization solution at 70 ◦C for 30 min followed by washing twice with MABT-buffer (100 mM maleic acid, 150 mM NaCl, 0.1% Tween-20). Slides were blocked with blocking solution (MABT + 1% blocking reagent: 1096176 Roche, Basel, Switzerland) for 1 h followed by incubation with anti-DIG antibody (11093274910 Roche, 1:4000 in blocking solution) over night at 4 ◦C. Slides were washed 5× 20 min with MABT-buffer and twice 10 min with NTMT-buffer (0.1 M NaCl, 0.1 M Tris HCl pH 9.5, 0.05 M MgCl2, 0.5% Tween-20). Signal detection was performed using 4-Nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP) according manufacturer’s instruction. To stop the reaction, slides were washed in 1 mM EDTA-PBS, mounted with a coverslip and analyzed using a Zeiss Axio Scope.A1 microscope. The same acquisition settings were used in all experiments and groups. 4.11. Further Analyses Further analyses such as the TUNEL assay, immunohistochemistry and western blot analysis were performed according to established standard protocols and are listed in the Supplementary Materials and Methods. 4.12. Statistical Analysis Values are presented as mean ± standard error of the mean (SEM). All statistical analyses were performed utilizing GraphPad Prism 5.03. One-way ANOVA and Bonferroni post hoc test was used to compare the groups. The value p < 0.05 was considered to be statistically significant. Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/7/2370/ s1. Author Contributions: N.H.: methodology, validation, formal analysis, writing—original draft preparation, writing—review and editing, visualization and conceptualization. U.S.: writing—original draft preparation and writing—review and editing. D.C.: methodology and validation. I.P.: writing—original draft preparation, writing—review and editing and resources. M.A.: resources and writing—review. M.H.H.S.: writing—review. R.H.W.F.: writing—review, resources and funding acquisition. C.R.: writing—review, funding acquisition and supervision. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by Novartis Pharma GmbH. Open Access Funding by the Publication Fund of the TU Dresden. Acknowledgments: The authors thank K. Pehlke, T. Schwalm and A. Neißer from the Department of Anatomy TU Dresden for their excellent technical assistance, A. Petzold and A. Dahl (Deep Sequencing Group SFB 655, Biotechnology Center, TU Dresden) for bioinformatics analysis, Silke Tulok for imaging assistance (Core Facility Cellular Imaging, TU Dresden), Hella Hartmann for assistance with laser capture microdissection (Light Microscopy Facility BIOTEC/CRTD, TU Dresden) V. Pawolski from the Department of Anatomy for comments on the manuscript and K. Engelmann (Department of Ophthalmology, Klinikum Chemnitz GmbH) for great collaboration. Conflicts of Interest: The authors declare that they have no competing interests. Abbreviations AMD Age-related macular degeneration BL Blue light C Control CCO Cytochrome c oxidase DCFDA 2′ ,7′ –dichlorofluorescin diacetate DEG Differentially expressed genes ETC Electron transport chain GCL Ganglion cell layerPDF Image | Photobiomodulation Mediates Neuroprotection against Retinal
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