Phalloidin ifluor 488 conjugate

Purified human Fbg, CitFbg, and hSAA1 (1 mL, 50 ng/mL) were coated on cover glasses overnight at 4 °C and washed with PBS three times. Then, the Fbg(hSAA1) and CitFbg(hSAA1) coated cover glasses were placed in the non-coated well. The PBS-coated cover glass was used as a control. Two mL of cell suspension (1 × 10⁶ cells /mL) was seeded into each well. PBS-0.75%BSA for MCF7 cells and 20% low glucose-DMEM-80% HBSS for MDAMB231 cells were used as cell suspension solutions for preventing nonspecific cell adhesion. Cells were cultured for 8 h to 3 days, and five images from the chamber were captured and viewed under an inverted microscope (CK40 Olympus, Tokyo, Japan) with a USB camera for Microscope Viewer Software v070817 (Shodensha, Osaka, Japan). On day 3, the cells were stained with phalloidin-iFluor 488 conjugate (1:1000, 20549, Cayman Chemical) and DAPI.

Antibodies used for western blotting and immunocytochemical analyses were as follows: anti-AKT (#9272), anti-YAP (#12395), anti-FOXO1 (#2880), anti-PTEN (#9559), anti-p53 (#2524), anti-HA (H3663), anti-β-actin (sc47778), anti-phosphorylated YAP (#13008), anti-phospho histone H3 (Ser10) (#9701), anti-phosphorylated AKT (threonine 308, #2965; serine 473, #9271), anti-phosphorylated FOXO1 (threonine 24 and 32, #9464), anti-cMYC (#5605), anti-E-cadherin (#3195), anti-N-cadherin (#14215) and anti-Merlin (#12888). All antibodies were purchased from Cell Signaling Technology, except for antibodies against HA-tag and β-actin, which were obtained from Sigma-Aldrich and Santa Cruz Biotechnology, respectively. The AKT inhibitor Mk2206 and SC66 from Selleckchem were used at final concentrations of 1 μM and 2 μg/ml respectively. The cMYC inhibitor 10058-F4 from Millipore was used at a final concentration of 64 μM. Phalloidin-iFluor 488 conjugate from Cayman Chemicals was used according to the manufacturer’s protocol.

VSMCs were fixed with 4% formalin for 10–15 min and permeabilized by 0.1% Triton X-100 (Fisher scientific, Waltham, MA) for 10 min. Non-specific binding sites were blocked with 0.1 mM glycine in 1% BSA for 30 min at room temperature. Next, cells were incubated with caveolin-1 primary antibody (1:200) for 2 h at room temperature, washed with 1X PBS, and incubated with secondary antibody Alexa Fluor® 594 (1:400; Abcam, Cambridge, United Kingdom) for 1 h at room temperature. Cells were co-stained with Phalloidin-iFluor™ 488 Conjugate (1:1000; Cayman chemical, Ann Arbor, MI) for 30 min. The stained cells were mounted in Permount™ Mounting Medium (Fisher scientific) and imaged using a laser scanning confocal microscope (Eclipse Ti, Nikon, Minato City, Tokyo, Japan).

After the respective incubation times, the cell-seeded scaffolds were washed one time with PBS and then fixated in 4% paraformaldehyde (methanol-free) diluted in PBS, for 1 h at room temperature. Subsequently, they were rinsed in PBS three times and stored in PBS containing antibiotics/antimycotic (ABM) (Pan Biotech, Aidenbach, Germany) at 4 °C, until staining.
The samples, prior to staining, were permeabilized with the washing buffer (PBS with 0.1% Triton X-100, Sigma-Aldrich, Saint Louis, MO, USA) for 5 min and rinsed three times in PBS. Phalloidin-ifluor 488 conjugate (Cayman chemical, Ann Arbor, MI, USA) with a 1000× dilution in 1% (w/v) BSA in PBS, was added to the samples to stain the actin filaments in the cell’s cytoskeleton, with an incubation time of 90 min at room temperature. The samples were rinsed three times in PBS and the nuclei were stained with DAPI (Carl Roth, Karlsruhe, Germany) for 15 min and then washed again in PBS for three times.

Fluorescent staining was carried out essentially as described previously^{23 (link)}. Coronal brain sections 20 µm in thickness were prepared using a cryostat and these were fixed with 4% PFA for 10 min at room temperature for fluorescent staining. The brain sections were incubated with DAPI and Phalloidin-iFluor 488 conjugate (Cayman Chemical, Ann Arbor, MI). The fluorescent images were captured using LSM 880 confocal microscope (Zeiss, Oberkochen, Germany). For HE and Nissl staining, mice were transcardially infused with 4% PFA and the brains were postfixed with the same fixative solution overnight. After dehydration with 30% sucrose, the coronal brain sections 20 µm in thickness were prepared using cryostat. For Nissl staining, the sections were stained with 0.1% cresyl violet solution for 20 min at 60 °C. The visible images were obtained using a BZ-9000 microscope (Keyence, Osaka, Japan).