Sab4200347

Proximities between actin and vimentin or nestin filaments were examined by Duolink in situ Proximity ligation assay (PLA) (Sigma-Aldrich). Cells were cultured and fixed with 4% paraformaldehyde for 15 min. After permeabilization with cold acetone for 1 min, the cells were washed with a blocking solution containing 0.4% Block Ace (DS Pharma Biomedical) in PBS, incubated with mouse monoclonal anti-actin antibodies (1:400) (AC-40, Sigma-Aldrich, St. Louis, MO, USA) and rabbit monoclonal anti-vimentin (1:500) (EPR3776, Abcam, Cambridge, UK) or rabbit monoclonal anti-nestin antibodies (1:1000) (SAB4200347, Sigma-Aldrich, St. Louis, MO, USA) diluted in 0.4% Block Ace for 1 h at room temperature. All other steps were performed according to manufacturer's instructions. To evaluate the number of signals from each PLA reaction, fluorescence spots on the basal membranes of 30 cells were counted.

Nanoneedles were fabricated from pyramidal silicon AFM cantilevers (ATEC-Cont, Nanosensors) and etched to a cylindrical shape of 200 nm in diameter and 10-15 µm in length using the FIB 31 (link). Spring constants (k = 0.1-0.4 N/m) were determined as described above. The silicon surface was cleaned with oxygen plasma in a JPA300 plasma asher (200 W, 5 min) (J-science, Kyoto, Japan) and treated with 1% HF for 1 min. After repeating the plasma (10 min) and 1% HF treatment, the nanoneedle was modified by physical adsorption of 50 μg/ml of ZZ-BNC at room temperature for 1 h; ZZ-BNC is bio-nanocapsule-based anchor to which the Fc domain of antibodies can bind 40 (link). Anti-vimentin (V6630; Sigma-Aldrich) or anti-nestin antibodies (SAB4200347; Sigma-Aldrich) (11.75 µg/ml each) were bound to the ZZ-domain of the ZZ-BNC by incubating in PBS overnight at 4°C. The antibody-immobilized nanoneedle was rinsed prior to use in cell fishing experiments. Force measurements were carried out using the AFM Nanowizard II with CellHesion. For cellular membrane penetration tests, nanoneedles were inserted into cells at an approach velocity of 10 µm/s with a set point of 200 nN, left to dwell within the cells for 60 s, and then evacuated at 10 µm/s. Ten different sites on each cell were targeted for insertion and three cells were tested for each cell type.