Treated cells (U937 macrophages or BMDMs) were fixed with 4% paraformaldehyde (Sigma, USA) and washed and permeabilized with 0.5% Triton X-100 and rinsed 3 times with PBS. Nonspecific binding sites were blocked by adding PBS with 1% BSA. The adhesion protein, vinculin, was labelled by incubation at 4 °C, overnight with mouse monoclonal anti-vinculin primary antibody, clone hVIN-1 (1:400, Sigma, USA). Cells were then washed and incubated for 1 h at room temperature in Alexa 488 conjugated anti-Mouse IgG as Secondary Antibody (1:1000, Invitrogen, USA). In separate samples, cellular α-tubulin and acetylated α-tubulin were labelled using rabbit polyclonal anti-α tubulin antibody (1:200, Abcam, UK) and mouse anti-acetylated tubulin, clone 611B-1 (1:2000; Sigma-Aldrich, USA) respectively with Alexa 488 secondary antibodies as above.
To simultaneously label the F-actin, the samples were incubated for 30 mins with 200 μl of rhodamine-conjugated phalloidin (Molecular Probes, USA) in a humidified chamber at room temperature in the dark. Cell nuclei were stained by incubation with 5 μg/ml DAPI (Dojindo, USA) followed by two PBS rinses. Slides were then visualized with a fluorescence microscope (SP2, Leica, Germany) with ×63 objective. Slides were also imaged on a microscope (710 ELYRA PS.1, Zeiss, Germany) with a ×63/1.4NA objective for structural illumination microscopy.
To simultaneously label the F-actin, the samples were incubated for 30 mins with 200 μl of rhodamine-conjugated phalloidin (Molecular Probes, USA) in a humidified chamber at room temperature in the dark. Cell nuclei were stained by incubation with 5 μg/ml DAPI (Dojindo, USA) followed by two PBS rinses. Slides were then visualized with a fluorescence microscope (SP2, Leica, Germany) with ×63 objective. Slides were also imaged on a microscope (710 ELYRA PS.1, Zeiss, Germany) with a ×63/1.4NA objective for structural illumination microscopy.