Las microscope software

To conduct wound healing assay, cells were seeded into 12-well plates and then incubated over 90% confluence. The plate was scratched with pipette tips and washed with PBS. Cells were incubated for 24 h with fresh DMEM medium containing either vehicle alone or ODZ10117. Digital images were obtained using the Leica Application Suite (LAS) Microscope Software (Leica Microsystems).
Invasion assay was performed using a Boyden chamber system (Neuro Probe, Gaithersburg, MD, USA). Growth factor reduced Matrigel (354230, BD Matrigel™, BD Biosciences) was diluted with serum free media with ratio of 1:3. Diluted Matrigel was transferred into 24-transwell (BD 24-well insert, 8 μm pore transparent PET filter) and incubated at least for 4 to 5 h for gelling at 37 °C. Cells in 100 μL DMEM containing 1% FBS were seeded in the upper chamber and incubated for 24 h in the presence of either vehicle alone or ODZ10117. The lower chamber was filled with 500 μL of 10% DMEM containing fibronectin (5 μg/mL, ECM001, Sigma-Aldrich). Matrigel containing upper chamber was rinsed with PBS, fixed, stained with Diff-Quik solution (Sysmex Corporation, Kobe, Japan), and subsequently rinsed with distilled water. The migrated cells were captured using the LAS Microscope Software (Leica Microsystems).

Tissue samples were fixed with 4% paraformaldehyde in 0.5 M phosphate buffer and embedded in paraffin. The paraffin blocks were cut in 4-μm-thick sections, mounted on glass slides, dewaxed, rehydrated with grade ethanol, and stained with hematoxylin and eosin (H&E, HT100132, Sigma Aldrich and S3309, Dako, Carpinteria, CA, USA). To perform immunohistochemical analysis, rehydrated slide sections were unmasked with 10 mM sodium citrate buffer, quenched endogenous peroxidase for 20 min in 3% hydrogen peroxide, blocked for 30 min in PBS containing 10% goat serum, and incubated at 4 °C for overnight with appropriate primary antibodies with 1:100 dilution. The sections were incubated with biotinylated secondary antibody (anti-rabbit for BA-1000, anti-mouse for BA-9200 and anti-goat for BA-5000, Vector Labs, Burlingame, CA, USA) compatible with the primary antibody for 30 min, subsequently incubated with streptavidin-HRP (550946, BD Pharmingen, San Jose, CA, USA) for 40 min, and stained with 3,3-diaminobenzidine (D22187, Invitrogen). Digital images were obtained using the LAS Microscope Software (Leica Microsystems, Wetzlar, Germany).