Sil300cs 30cc

The VSMCs were incubated on glass-bottom culture dishes (No. 1, Matsunami Glass Industry) coated with 5% fibronectin (F1141, Sigma-Aldrich) in phosphate-buffered saline (PBS) for 60 min. The cells were imaged under an inverted fluorescence microscope (IX71, Olympus) equipped with a birefringence imaging system (Abrio-LS, CRi)^{21 (link),40 (link)} to analyse the retardation in samples. Because the retardation in background areas changed with time, a background image was taken just before imaging a cell. Cell retardation images were processed by subtracting background retardation from cell retardation. The retardation image was captured through an objective lens of 60× (UPLSAPO60XW, NA = 1.20, Olympus; UPLSAPO60XS2, NA = 1.30, Olympus) or 30× (UPLSAPO30XS, NA = 1.05, Olympus) with silicone immersion oil (SIL300CS-30CC, Olympus). DIC images of the cells were also obtained.

HEK293T cells or hippocampal neurons grown on coverslips were washed with PBS and fixed at room temperature using 4% paraformaldehyde (PFA) in PBS for 10 min or 4% PFA and 4% sucrose in PBS for 15 min, respectively. For immunocytochemistry of endogenous PSD-95, hippocampal neurons were incubated with blocking solution (2% normal goat serum (NGS), 0.2% Triton X-100, PBS) for 1 h at room temperature. Cells were washed with PBS twice and then incubated in antibody solution (2% NGS, 0.2% Triton X-100, PBS) with anti-PSD-95 antibody (Millipore, MABN68) for overnight at 4°C. Cells were washed with PBS 3 times and incubated with antibody solution with Alexa Fluor 568-conjugated anti-mouse IgG antibody (Molecular Probes, A-11019) for 1 h at room temperature. Cells were washed with PBS 4 times and embedded with Mounting Medium (Vectashield, H-1000). Cells were observed using FV3000 confocal microscopy (Olympus) equipped with a 40× objective lens (Olympus, N2246700), a 60× objective lens (Olympus, N1480700), or a 100× objective lens (Olympus, N5203100). Immersion oil (Olympus, IMMOIL-F30CC) or silicone immersion oil (Olympus, SIL300CS-30CC) were used for 60× and 100× lenses, respectively.