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Fig. 5. Induction of extracellular matrix TSP1 and versican by a TLR3 agonist. (A) HASMC were incubated in control medium or medium containing 5 µg ml–1 poly I:C for 4 hours or 15 hours, followed by fixation and double immunofluorescent staining without permeabilization using antibodies to versican (green) and to TSP1 (red). Nuclei were counterstained with Hoechst 33258 (blue). Note that co-localization of versican and TSP1 appears as yellow close to the cell surface after treatment of HASMC with poly I:C in a time-dependent manner. Bars, 50 µm. Results are representative of at least three independent experiments. (B,C) Confocal images showing extracellular localization of TSP1 and versican after stimulation with poly I:C. HASMC were cultured without treatment (B, left) or treated with 5 µg ml–1 poly I:C for 15 hours (B, right and C) and cells were fixed, permeabilized and double stained for versican (green) and TSP1 (red). Stack of images (C, 1-6) through the Z-plane from inside (1) of the cells to the cell surface was generated using confocal microscopy. Nuclei were counterstained with DAPI (blue). Bar, 10 µm. (D) Confocal 3D reconstruction of 18 hour poly I:C-stimulated HASMC stained without permeabilization for versican (green) and TSP1 (red). Bar, 10 µm.
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