Flowview software

MCF7 cells expressing mCherry-vimentin or its mutants were seeded on glass-bottomed dishes. FRAP measurements were performed under a confocal microscope equipped with two simultaneous laser scanners (FV1000, Olympus, Tokyo, Japan) in a 37 °C and 5% CO₂ environment. A 50-mW 405-nm laser set at 100% was used through one scanner for 1 s to photobleach a 3-μm wide band across the cells, and the fluorescence images of mCherry-vimentin (543 nm laser excitation) were obtained over time for 60 min through the second scanner. For the FRAP analysis, the recovery of the relative fluorescence ratios in the cytosol was normalized to the same regions before photobleaching after background subtraction and analyzed using FLOWVIEW software (Olympus).

The embryos were transferred into HEPES-buffered CZB covered with mineral oil. The stage of the confocal microscope was warmed with a thermo plate (Tokai Hit, Shizuoka, Japan). The region of interest (ROI), reference region (ref) and background (back) were set using Flowview software (Olympus). The areas of these regions were set at 40 × 40 pixels (7.6 μm²). Three pictures were taken at 1.6 s intervals (free run setting), after which the ROI was photobleached with the 110 μW laser at 488 (FV1000) or 477 (FV1200) nm for 5 s. A total of 9 pictures was taken at 1.6-s intervals, and the intensities of the ROI, ref, and back were measured in each picture. To reduce the toxicity to zygotes, the observation time (bleaching and imaging time) was set at 23 s, which is shorter than that used in our previous study (150 s) [13 (link)]. For measuring intensity, the 488 or 477-nm laser output was set at 15 μW. The recovery rate and mobile fraction were calculated as described previously [13 (link)].

For IHC assessment, a tissue micro-array was constructed using cores of 0.6 mm in diameter. Three zones were used for each tumor: intratumoral, invasion front, and peritumoral. Three cores were used for each zone. Peritumoral zones were not obtained for two tumors. Sequential 3-μm sections were cut and colored by HES or used for immunohistochemistry.
IHC was also performed on tissue sections from formalin-fixed paraffin-embedded tissue blocks from WT and OSM-KO mice. Serial sections of 3 μm were cut. Immunohistochemistry was performed using a BenchMark automated staining system (Ventana Medical System, Tucson, AZ) for CD3 (Dako, A0452, 1:200), CD68 (Ventana, 790-2931, ready to use), and MPO (Dako, IS511, ready to use). Images were taken using a camera fixed to the microscope (Leica Application suite, version 4.4). Quantification of inflammatory cells was performed using assisted counting software (Visilog Noesis).
For immunofluorescence studies, tumor sections were fixed in acetone/methanol (20/80) and stained with anti-OSM (R&D system, MAB4951, 1:50), followed by staining with a goat anti-rat-IgGAF555 secondary antibody (Thermofisher, A-21434, 1:100). Cell nuclei were stained with TOPRO (Invitrogen). Image acquisition was performed with an Olympus FV1000 confocal microscope using FlowView software (ImageUP platform, University of Poitiers).