Anti psa

Immunocytochemistry of C2C12 myoblasts and myotubes was performed as described previously (Osana, Kitajima, et al., 2020; Osana, Murayama, et al., 2020). Samples were incubated with primary antibodies at 4°C overnight following blocking/permeabilization with phosphate‐buffered saline containing 0.05% Triton X‐100% and 5% goat serum for 60 min at room temperature. The immunocytochemistry of anti‐PSA (1:100; Santa Cruz Biotechnology), anti‐Ki67 (1:500; Abcam), anti‐MyHC (1:200; Developmental Studies Hybridoma Bank), anti‐α‐actinin (1:200; Abcam), anti‐β‐actin (1:2000; Cell Signaling Technology), and nuclei was visualized using appropriate species‐specific Alexa Fluor 488‐ or Alexa Fluor 555‐conjugated secondary antibodies and Hoechst 33342 (Thermo Fisher Scientific). Samples were then examined using an Olympus fluorescence microscope (Olympus Corporation). The relative ratio of Ki67‐positive cells was calculated by dividing the number of Ki67‐positive cells by the total number of nuclei, namely the total number of cells. The differentiation index was calculated by dividing the number of nuclei in myotubes (MyHC‐positive elongated cells) by the total number of nuclei. The length of myotubes and the aspect ratio, which represents the ratio of width to length, were measured using ImageJ Fiji software (Schneider et al., 2012).

Cells isolated or transfected after 24 hours were quickly washed once with PBS, fixed with 2% PFA, and blocked with 2% goat serum. Cells were then stained with anti-PSA, anti-Twist, anti-E-cadherin, anti-vimentin, or anti-survivin antibody (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight and then visualized via incubation with AF488 or AF594 secondary antibody for 1 hour at room temperature. Cells were imaged using an LSM 510 confocal microscope (Zeiss, Oberkochen, Germany) as described previously.^{23 (link)}