Lsm 710 confocal laser system

A Zeiss LSM 710 confocal laser system was used for the investigations and the 3D images were captured and analyzed by the ZEN 2012 SP1 software (black edition). The technical specifications of the used equipment are: diode laser (405 nm), Ar-laser (458, 488, 514 nm), diode pumped solid state laser (561 nm) and HeNe-laser (633 nm), AxioObserver Z1 inverted microscope, 40× apochromatic objective (numerical aperture 1.4) and the FS49, FS38 and FS15 filters. To capture the autofluorescence of the native powders, the emission was measured at wavelength between 405–633 nm. In order to acquire the images, the microparticles were stained with two dyes: 4′,6-diamidino-2-phenylindole (1 μg/mL) and Red Congo (40 μM), in a ratio 3:1:1. To increase the signal-to-noise ratio, the frame average of eight scans was used.

A549 cells were seeded on glass slides and treated as indicated for 24 h. The slides were washed with PBS, followed by fixation with 4% paraformaldehyde for 10 min and permeabilization with 0.1% Triton X-100 in PBS. The cells were blocked with 2% BSA at room temperature for 1 h and incubated with primary antibodies (E-cadherin, 1:200; fibronectin, 1:200; α-SMA, 1:200) at 4 °C overnight. After incubation, samples were washed three times with PBS and incubated with corresponding secondary antibodies (1:200, Beyotime Institute of Biotechnology, Haimen, China). The nuclei were stained with DAPI. Samples were washed three times with PBS, coverslips mounted in 90% glycerol in PBS, and fluorescence detected using a Zeiss LSM 710 confocal laser system.