Dm2000 optical microscope

For each mouse (n=6), tumor size was assessed thrice per week by caliper measurement. Tumor volume was calculated as follows:
$\text{Volume}=\mathrm{D}\times {\mathrm{d}}^2\times \mathrm{\pi }/6$ where D and d are the longer and the shorter diameters, respectively. After ex vivo OI acquisitions, explanted organs and sc tumor masses were fixed in 10% buffered-formalin solution and embedded in paraffin for histological analysis, or embedded in OCT (optimal cutting temperature) compound embedding medium (Miles, Inc., Diagnostics Division, Elkhart, IN, USA) and snap-frozen at −80°C for fluorescence microscopy. For confocal microscopy analysis, sections of 30 μm thickness were cut from frozen organs with a cryostat at −20°C and analyzed using confocal microscope Eclipse te300 (Nikon Corporation, Tokyo, Japan). Immunohistochemical analysis of the tumoral masses were performed with hematoxylin and eosin to evaluate the tissue morphology, and with the avidin–biotin–peroxidase complex to localize CD5, CD20, CD45, and CD79a antigens.25 (link) The slides were examined under a Leica DM2000 optical microscope (Leica Microsystems).

Migration ability of cancer cells was assessed by wound-healing and Transwell assays. In the wound-healing assay, the cells were cultured into 6-well plates at a density of 2×10⁵ cells/well. When the cells grew to 90% confluence, they were incubated with mitomycin-C (10 µg/ml) for 1 h to suppress proliferation and then starved in serum-free medium for 24 h. An artificial wound was created by scraping the cell confluent monolayer with a 10-µl pipette tip. The migration gap of the wound was assessed after 48 h. The migration and invasion potential was also evaluated by Transwell assay. The cells were treated with mitomycin-C (10 µg/ml) for 1 h at 37°C in serum-free medium and then plated into the upper chamber. For the migration assay the cells were seeded with a density of 2×10⁴ cells/insert, and for the invasion assay cells are seeded into the top chamber coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) at a density of 4×10⁴ cells/insert. Then DMEM with 10% fetal bovine serum (FBS) was added into the bottom chamber. After incubation for 24–48 h, the cells adhering to the lower membrane of the inserts were stained with 0.1% crystal violet and observed by a Leica DM2000 optical microscope (Leica Microsystems) then counted by ImageJ k1.45 software (National Institutes of Health). All the experiments were replicated in triplicate.