Alexa fluor plus 555 secondary antibody

NF-MP or FF-MP were fixed with 4% paraformaldehyde for 30 min and permeated with 0.3% Triton-100 for 10 min. After being blocked with 5% goat serum (Gibco, #16210064) for 4 h at room temperature, the NF-MP or FF-MP were stained with the anti-vinculin antibody (1:150, Abcam, ab129002), ani-ROCK1 antibody (1:200, Abcam, ab45171) or anti-YAP1 antibody (1:500, Abcam, ab39361), or anti-Runx2 antibody (1:150, Abcam, ab23981) overnight together with CF633 phalloidin (10 U/mL, Biotium, 00046) at 4 °C. The samples were stained with Alexa Fluor Plus 555 secondary antibody (1:200, Invitrogen, A32732) for 2 h at room temperature, followed by 1 μg/mL Hoechst 33342 for 20 min. The NF-MP or FF-MP was placed on a glass slide and mounted for observation under a confocal laser scan microscope (TCS SP5, Leica, Buffalo). For cellular processes analysis, we consider a cytoplasmic extension as a cellular process when the distance between its distal end and the cell body is more than 5 μm.

For actin staining, the NF-MT samples were permeated with 0.3% Triton X-100 for 10 min, blocked with 5% goat serum for 30 min at room temperature, and incubated with 10 U/mL CF633 phalloidin (Biotium, 00046) for 60 min at 37 °C. For nuclear staining, the samples were immersed into 1 μg/mL Hoechst 33342 (Thermo Scientific, 62249) for 10 min at room temperature. For immunofluorescence staining, the NF-MT samples were permeated with Triton X-100 for 10 min and blocked with 5% goat serum to prevent nonspecific staining for 4 h at room temperature. Afterward, the samples were incubated with primary antibodies that included an anti-integrin beta-1 antibody (1:1000, Abcam 179471), anti-vinculin antibody (1:150, Abcam 129002), anti-β tubulin antibody (1:400, Abcam 52901), anti-vimentin antibody (1: 500, Abcam 92547), anti-collagen I antibody (1:200, Abcam 6308), and anti-Golgi antibody (1:200, Invitrogen PA3–910). The samples were then washed with PBS and incubated with an Alexa Fluor Plus 555 secondary antibody (1:200, Invitrogen, A32732) with 1% goat serum (1:200, Invitrogen, A32732) for 2 h at room temperature.