Dp2 bsw software

Free-floating coronal sections (40-µm thick) were washed in phosphate-buffered saline at room temperature and blocked for 1 h in 10% serum and 0.03% Triton X-100 in phosphate-buffered saline. Sections were incubated with primary antibody in 2% serum and 0.03% Triton-X in phosphate-buffered saline overnight at 4°C. Primary antibodies to tyrosine hydroxylase (AB152; Millipore; 1:500), NeuN (MAB377; Millipore; 1:200), GFAP (1:400; 13-0300; Invitrogen), and hSNCA (MAB5320; Millipore; 1:400) were incubated at 2% serum and 0.03% Triton-100 in phosphate-buffered saline for 1 h at room temperature. Sections were stained with Alexa Fluor fluorescent secondary antibodies matched to the host primary (Alexa Fluor 488, 555, 568, 633) (1:400; Jackson Immunoresearch, West Grove, PA, USA) in 2% serum and 0.03% Triton-X at room temperature for 1 h. All sections were mounted onto Superfrost Plus slides (Fischer Scientific, Pittsburgh, PA, USA) and cover slipped with Fluoroshield with DAPI (F6057; Sigma). Images were captured on Leica Leitz Digital Module R fluorescent microscope (Leica Microsystems, Buffalo Grove, IL, USA) connected to a Olympus DP72 camera (Olympus, Melville, NY, USA) using the Olympus DP2-BSW software (Olympus). Immunopositive cells were counted per field of view at 40× using ImageJ by two blinded investigators. Cell counts were compared via t-tests.

Interphase FISH was performed on TMAs of 150 cases from the same cohort as previously described [17] (link). The Vysis LSI MYC dual color, break apart rearrangement probe, the Vysis LSI BCL6 dual color, break apart rearrangement probe and the Vysis LSI IGH/BCL2 dual fusion translocation probe (Abbott Molecular, Abbott Park, IL) were used. FISH signals were analyzed using a fluorescence microscope (Olympus BX51, Tokyo, Japan) equipped with a DP72 camera and DP2-BSW software (Olympus, Tokyo, Japan). Patient cases with break-apart signals in >10% of nuclei were considered positive for the presence of a translocation. The signal distribution was evaluated by two independent observers (Dong-Lan Luo and Jie Cheng). In case of discordant results between the two observers, a third investigator (Jie Xu) was involved.

The cell migration assay was performed using trans-well plates with 8.0-μm polycarbonate membranes (Corning). 1 × 10⁵ cancer cells were seeded onto the upper chamber. The cells were tested for the migration toward the lower chamber during stimulation by serum with or without S100A4 (2 μg/ml) or S100A4 neutralizing antibodies (4A, 30 μg/ml). After 16 h, migrated cells were fixed with fixing solution, followed by crystal violet staining for visualization with a microscope (Olympus BX51, 20x objective lenses, DP72 camera, DP2-BSW software (version 2.2)).

BMMs were prepared from 5-week-old female ICR mice as previously described [29 (link)]. Pre-osteoclasts were generated by culturing BMMs with M-CSF (30 ng/mL) and RANKL (50 ng/mL) for 36 to 48 h. Then, mature osteoclast formation was induced by incubating pre-osteoclasts with M-CSF (30 ng/mL) and conditioned medium from cancer cells. Multinucleated TRAP+ cells usually formed within 24 to 48 h after conditioned medium treatment. TRAP+ cells with 3 or more nuclei were visualized under a microscope and considered to be mature osteoclasts (Olympus BX51, 20x objective lenses, DP72 camera, DP2-BSW software (version 2.2)).

The proliferation of recovered MSCs from PEG 400*, PEG 600*, PEG 1 K, PEG 1.5 K, and PEG 5 K solutions was assayed on days 1, 3, and 5 using CCK-8. PEG 400* and PEG 600* indicate that cells were preincubated for 2 h at 37 °C before cryopreservation. The recovered live cells (5.0 × 10⁴ cells/well) were seeded to the 24-well plate with the growth media. The proliferation of MSCs recovered from cryopreservation using the traditional DMSO 10% system was also compared as a control. The cell images were obtained at 1, 3, and 5 days during the cell proliferation using the live/dead assay kit. Briefly, the cells were treated for 30 min with ethidium homodimer-1 (2.0 μM) and calcein AM (2.0 μM) solution. The fluorescence images were obtained using an Olympus IX71 fluorescence microscope and Olympus DP2-BSW software. At 1, 3, and 5 days of proliferation, the growth medium was replaced with CCK-8 solution (10% in DMEM containing 1.0% PS), and incubated for 150 min at 37 °C. The absorbance was measured at 450 nm relative to 655 nm using a microplate reader (iMARK, Bio-Rad, USA) for a quantitative assay for cell proliferation. 100% was assigned to 0 days (3 h) of data.

The multilineage differentiation potentials of cryopreserved MSCs were evaluated for osteo-, chondro-, and adipogenic differentiation. The cells were seeded in 24-well plate (5.0 × 10⁴ cells/well). After confluence, the cells were treated with specific osteo-, chondro-, and adipogenic induction media [24 (link)]. The differentiation media was replaced every 2 days with fresh media for two weeks. On the last day of differentiation, the cells were fixed with 4% formaldehyde and washed with PBS. Thereafter, the fixed cells were stained with alizarin red, alcian blue, and oil red O for osteo-, chondro-, and adipogenic differentiation, respectively. The images were captured by an Olympus IX71 fluorescence microscope using Olympus DP2-BSW software.