Acti stain 555 phalloidin phdh1 a

Caco-2 cells were cultured in 8-chamber slides and fixed with 4% paraformaldehyde at room temperature for 5 min. Then, 50 μL of microbeads was mixed with 450 μL of PBS (i.e., 10-fold dilution). The suspension (100 μL) was added to each well and incubated at room temperature for 1 h. After washing three times with 100 μL of PBS, the cells were stained with Acti-stain 555 phalloidin (PHDH1-A; Cytoskeleton, Inc., Denver, CO, USA) to visualize actin filaments. Fluorescence images were obtained using a Pulse-SIM BZ-X700 microscope equipped with the Hybrid Cell Count BZ-H3C software (Keyence, Osaka, Japan).
For scanning electron microscopy (SEM), the slide was mounted on aluminum stubs with double-sided carbon tape (Nisshin EM, Tokyo, Japan) and coated with platinum-palladium using E-1030 Ion sputter (Hitachi, Tokyo, Japan). Images were acquired using a Hitachi S-4700 FE-SEM (accelerating voltage, 10 kV).

Immunohistochemical analysis was performed as previously described (Sakagami et al. 2021 (link)). The following primary antibodies were used: mouse anti-dSap4 antibody (DSAP47-1, Developmental Studies Hybridoma Bank, Iowa, USA) at a 1:20 dilution and mouse anti-α-tubulin antibody (DM1A, Thermo Fisher Scientific, Tokyo, Japan) at a 1:200 dilution. The following secondary antibodies were used: goat anti-mouse IgG antibody, Alexa Fluor 488 (A11001, Thermos Fisher Scientific, Tokyo, Japan), at a 1:200 dilution. Cytological staining for microvilli and muscles was done with Acti-stain 555 phalloidin (PHDH1-A, Cytoskeleton Inc., Denver, USA) at a 1:200 dilution. Fluorescent images were taken using a confocal laser scanning microscope system (FV1200, Olympus, Tokyo, Japan). Depth coding, stereo pair, and orthogonal images were reconstructed from confocal image stacks using LSM 510 software (version 3.2; Carl Zeiss, Germany) and Imaris software (version 9.3; Oxford Instruments, Oxfordshire, UK).