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. 2016 Nov 3;6:36418. doi: 10.1038/srep36418

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Copyright © 2016, The Author(s)

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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hBMSC were cultured on TCPS. From 24 h after seeding, hBMSC were treated with 5 μg AlexaFluor488-FN/mL (green fluorescence), 45 μg plasma-FN/mL and 200 μg ATTO565-sHA1/mL (red fluorescence) simultaneously. After further 48 h cells were fixed, stained with DAPI and embedded in Mowiol 4-88 as described before. Yellow fluorescence indicated overlaid structures of AlexaFluor488-FN and ATTO565-sHA1 (left panel). To identify and quantify colocalization, threshold was set using Otsu’s method and images were visualized in false colors (right panel; signal in white, background in black) and subjected to JACoP Plugin in Fiji Software. Colocalized structures are colored in white (right panel). Scale bar 5 μm. The data of colocalization analysis of 15 randomly chosen images (values as mean ± SEM (standard error of the mean)) are summarized underneath.