Fluoview fv10i doc

Immunocytochemistry of 293T cells was performed essentially as previously described [22 (link)]. In brief, cells centrifuged onto slides were fixed in 4% paraformaldehyde and permeabilized in 0.1% Triton X-100 for 10 min each. Slides were then incubated stepwise in blocking solution (5% goat serum in PBS), the primary antibody (1:200 in the blocking solution), and then the secondary antibody (1:1000 in the blocking solution) for 1 h each at room temperature. The aggresomes were visualized using PROTEOSTAT Aggresome Detection Kit (Enzo Life Sciences), and the nucleus was counterstained using ProLong Gold antifade reagent with DAPI (Invitrogen). Images were acquired using a confocal microscope (Fluoview FV10i-DOC, Olympus, Tokyo, Japan) and analyzed with FV10 ASW10 software (Olympus).

Cells were rotated under serum-free conditions for 30 min, and stimulated with 10% FCS followed by incubation for 0–60 min. Cells were fixed in 4% paraformaldehyde for 10 min at room temperature. After being washed with PBS, nonspecific binding was blocked with 2.5% normal donkey serum and 5% normal goat serum in PBS for 30 min at room temperature. Cells were incubated with biotin-conjugated anti-human CD29 antibody and anti-caveolin-1 antibody in PBS for 45 min at room temperature, then with streptavidin-Alexa 488 and anti-rabbit IgG-Alexa 594 in PBS for 30 min at room temperature. The resulting staining patterns were imaged using a confocal microscope (Fluoview FV10i-DOC, Olympus, Tokyo, Japan).