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. 2016 Dec 16;6:39112. doi: 10.1038/srep39112

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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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(a) OA cartilage sections were processed with antibodies against Gal-3 (red) – followed by immunofluorescence detection using AlexaFluor555-labelled second-step antibodies – together with DAPI (blue) prior to analysis using laser scanning microscopy. Differential interference contrast (DIC) imaging was included. Scale bar: 20 μm. A series of eight images was recorded at 1 μm intervals to create a stack in the Z axis. Shown is the projection from the Z stack generated using ZEN software. (b) OA cartilage sections were processed with Gal-3-FITC (green) together with DAPI (blue) prior to analysis using laser scanning microscopy. DIC imaging was included. Scale bar: 20 μm. A stack in the Z axis was created, based on eight serial microphotographs. Shown is the projection from the Z stack established using ZEN software. (c) Cultured OA chondrocytes were trypsinised and resuspended prior to labelling with Alexa-Fluor555-labelled Gal-3 (Gal-3-555) (red) and DAPI (blue) at 4 °C for 10 minutes in presence or absence of 0.1 M β-lactose. After 10 minutes of incubation, cells were washed and analysed using laser-scanning microscopy, with the focus plane set to the centre of cells. Scale bars: 20 μm. (d,e) Cultured OA chondrocytes were trypsinised and resuspended prior to labelling with (d) Gal-3-555 (red) and AlexaFluor488-labelled (Gal-3tr-488) (green) or (e) Gal-3-555 (red) and Gal-1-488 (green) at 4 °C for 10 minutes. After 10 minutes of incubation, cells were washed and analysed using laser-scanning microscopy, with the focus plane set to the centre of cells. Co-localization is presented in separate and overlay images. Arrows mark the staining of cell nuclei of chondrocytes, whose cell membranes have lost their integrity, by (d,e) Gal-3-555 (red), but not by (d) Gal-3tr-488 (green) or (e) Gal-1-488 (green). Shown are the results from chondrocytes of one patient, representative for three independent experiments (n = 3 patients). Scale bars: 20 μm.