Phalloidin tetramethylrhodamine b isothiocyanate phalloidin tritc

For direct fluorescent staining, samples were treated first with the IC₉₀ of the statin. After 24 h of incubation, cells were fixed with formaldehyde and deposited on a pre-coated coverslip. Later, cells were treated with Triton (0.1%) for 30 min followed by phalloidin-tetramethylrhodamine B isothiocyanate (phalloidin-TRITC; Sigma-Aldrich, Madrid, Spain) for another 30 min at room temperature. Finally, cells were washed with PBS. Cells were examined by Z-stack imaging with a 100× objective of EVOS FL Cell Imaging System AMF4300, Life Technologies, Carlsbad, CA, USA, at λexc = 540 nm and λem = 570 nm. As a positive control of actin depolymerization, methyl-β-cyclodextrin (BioReagent, Sigma-Aldrich, Madrid, Spain) was used [22 (link)]. Non-treated cells, containing 0.1% of DMSO, were considered as the negative control.

The fibrous PLA-based scaffolds and implants were analyzed with the Phenom scanning electron microscope (SEM) supported with image software (FEI, USA). All SEM samples were incubated in 0.5 % OsO₄ for 1 h at 4 °C, then dehydrated/desiccated with anhydrous ethanol, next automatically critical point dried (Leica EM CPD300, Germany), and finally coated with 15-nm layer of gold (K550 Emitech, USA) prior to SEM analysis.
Live/Dead Cell Staining Kit (Sigma Aldrich, USA) was utilized for simultaneous fluorescence staining and visualization (Nikon Eclipse Ti microscope) of viable and dead cells.
The implants were also analyzed with the Zeiss Axiovert 100 M confocal laser scanning microscope (CLSM) supported with LSM 510 META software (Carl Zeiss Jena GmbH, Germany). CP5 cells were stained (500 ng mL⁻¹) with phalloidin—tetramethylrhodamine B isothiocyanate (phalloidin-TRITC; Sigma, USA) to identify filamentous actin and with DRAQ5™ (Biostatus, UK) intercalating anthraquinone to visualize chromatin. Each imaging analysis was performed at least three times using material collected during three independent experiments.

For immunofluorescence analysis, cells were cytospun onto slides using a Shandon cytospin III cytocentrifuge and then fixed with 4% paraformaldehyde in phosphate‐buffered saline (PBS) at room temperature for 10 min and perforated with 1% Triton X‐100 in PBS for 4 min. After treatment with 1% bovine serum albumin in PBS for 30 min, the cells were incubated at 4°C overnight with a rabbit anti‐h2‐calponin antibody RAH2 or normal rabbit serum (NS) at 1:200 dilution. A second antibody, FITC‐conjugated anti‐rabbit IgG (Sigma) was utilized at a 1:200 dilution and incubated at room temperature for 60 min in the dark. Filamentous actin was stained with phalloidin–tetramethylrhodamine B isothiocyanate (phalloidin‐TRITC, Sigma) at 1:100 dilution. Coverslips were mounted onto slides with ProLong antifade with DAPI (for nuclear staining) mounting medium (Invitrogen, Carmillo, CA) and air‐dried in the dark for 24 h, sealed with varnish. Cells were examined with a Zeiss Axioimager Z1 fluorescence microscope (Carl Zeiss, Oberkochen, Germany) and a 63× , numerical aperture 1.4 oil immersion objective lens. Images were acquired using a Zeiss AxioCam MRm monochrome cooled‐CCD camera and analyzed using Axiovision version 4.8 software (Carl Zeiss).