Figure 3

Immunofluorescent confocal microscopic analysis of Tensin3 and ExoC6B in proband and control fibroblasts. A: Live cells were imaged using phase contrast microscopy; scale bar represents 63 μm. B: Cells were fixed, permeabilised and stained using specific antibodies: anti-Tensin3 in-house antibody no. 1 (recognising a C-terminal sequence; green fluorescence; top row), anti-Tensin3 in-house antibody no. 2 (recognising an internal sequence; green fluorescence; middle row), and a commercial goat anti-ExoC6B antibody (red fluorescence; bottom row). The secondary antibody was coupled to Alexa Fluor® 647 fluorochrome. Stained cells were imaged under a confocal microscope as described. Arrows indicate Tensin3 immunoreactivity in actin filamentous structures. Negative control images were also taken of cells not incubated with primary antibody (third image each row). Scale bar represents 20 μm.