Leica software

HLE B-3 cells and BMs grown on 24-well plates following various treatments were fixed with 4% paraformaldehyde solution containing 0.18% Triton X-100 for 30 min at 4°C. After rinsing thrice PBS, specimens were incubated with PBS containing 2% goat serum (Shang hai yuan mu, Shanghai, China) for 1 h at 37°C. Fixed cells or BMs were then incubated for 1 h at 37°C with the rabbit pAbs against p21 (1:500), LMα4 (1:100), LMβ3 (1:50) and MMP-9 (1:100; 10375-2-AP, Proteintech). After washing thrice with PBS, cells or BMs were incubated with FITC-anti-rabbit IgG (1:250, A11008, Life Technologies, New York, USA) for 30 min at room temperature. Next, the cells were washed thrice with PBS and incubated with DAPI (1:5000, C0060, Solarbio, Beijing, China) in PBS for 2 min at room temperature. However, this step was not performed for BMs. Finally, the cells or BMs were visualized using a Leica DMRA immunofluorescence microscope and Leica software (Leica Microsystems, Wetzlar, Germany).

The substrates were then washed three times with PBS. Then cell actin cytoskeleton was stained with phalloidin FITC Atto 488 (50 μL at 20 μg/mL in PBS in each well, contact time 30 min) (Sigma-Aldrich, Milan, Italy). Subsequently, after three PBS washes, the cell nuclei were stained with Hoechst 33258 (100 μL of solution at 1:10,000 dilution in PBS per each well, contact time 10 min in the dark) (Sigma-Aldrich, Milan, Italy), for 10 min. After three further PBS washes, the scaffolds were mounted on glass slides, covered using coverslips and analyzed using CLSM (Leica TCS SP2, Leica Microsystems, Milan, Italy) at λ_ex = 346 nm and λ_em = 460 nm for Hoechst 33258 and λ_ex = 501 nm and λ_em = 523 nm for phalloidin FITC. The acquired images were processed by means of Leica software (Leica Microsystem, Milan, Italy).

Imaging of the samples, either HUVECs or HEK-293 T cells, was co-transfected with plasmids carrying AcGFP or DsRed fluorescence, and carried out on a Leica SR-GSD microscope. Images were taken in TIRF (total internal reflection fluorescence) mode at a rate of 100 frames per s. The setup consisted of the following components: an inverted microscope (DMI6000 B, Leica Microsystems), a 1.47-NA TIRF objective, a 488-nm fiber laser (for green fluorescence), a 532-nm fiber laser (for red fluorescence), and an EMCCD camera. The super-resolution images were taken in TIRF mode at 100 frames per s and reconstructed from a series of ~ 5,100 images, giving a total measurement time of about 1 min for each color channel. Pixel size in the image was 100 nm. Quantitative colocalization ratio analysis of multicolor fluorescence images was performed with the Imaris software suite version 7.6 (Bitplane, Zurich, Switzerland).
Each cell sample was placed under a coverslip, sealed, and examined under 100× objective magnification using a Leica TCS-SP5-MP-SMD confocal system (Leica Microsystems). Images of the two different fluorochromes were collected at 1-µm-thick optical sections using Leica software (Leica Microsystems). The colocalization analysis was done with the Imaris software suite, version 7.6.