Ultravision protein block solution

NRP2 or NT siRNA transfected HUH6 cells were grown in 2-well chamber slides coated with collagen I for 72 h. Fixation was performed with 4% paraformaldehyde. Following, 0.1% Triton-X was used for permeabilization. Non-specific binding was blocked with UltraVision Protein Block solution (Thermo Scientific, Fremont, CA, USA). F-actin staining was carried out with phalloidin-FITC (at dilution 1:500; P5282, Sigma Aldrich) at room temperature for 1 h. Images were captured with Zeiss Axio Imager M2 (objective: EC Plan Neofluar 40 × /0.75 Ph 2 M27) (Carl-Zeiss, Oberkochen, Germany).

Cells were fixed on Ibidi eight-well μ slides with 3.8% paraformaldehyde at room temperature for 15 min and washed three times with PBS. For the nuclear epitopes, the cells were permeabilized using 0.5% Triton X-100-PBS at room temperature for 7 min. The cells were washed once with PBS, and unspecific binding of antibodies was blocked by Ultravision Protein Block solution (Thermo Fisher Scientific) by a 10-min incubation at room temperature. Primary antibodies were diluted in washing buffer (0.1% Tween20-PBS) and incubated at 4°C overnight. Excess primary antibody solutions were removed, and the cells were washed three times with washing buffer. The secondary antibodies were diluted 1:1,000 in washing buffer and incubated at room temperature for 30 min. The samples were washed three times with washing buffer, and nuclei were counterstained with DAPI, diluted 1:1,000 in washing buffer. The samples were washed once and kept in PBS for imaging. Protocols of western blotting and annexin V staining are provided in supplemental experimental procedures. All the antibodies used in this study are listed in the key resources table.