Leica dfc420c

Tissue samples prior to therapy were available for 9 out of 20 metastatic melanoma patients from cohort one. The samples were taken for diagnostic histological examination and were formalin-fixed and paraffin-embedded in the Department of Pathology of the Kantonsspital St. Gallen using the standard processing protocols. Four-micron-thick serial sections were then cut using a rotary microtome. Single epitope enzymatic immunohistochemistry on FFPE tissue was performed on serial sections to assess the % of tumor tissue expressing gp100 and MelanA/MART1 using a Leica BOND MAX automated immunostainer and the following antibodies: monoclonal mouse anti-human MelanA (Dako, catalog number M7196, clone A103, dilution 1:150, HIER - pH 9/20 min/95 °C, incubation for 15 min), and monoclonal mouse anti-human Melanosome (Dako, catalog number M0634, clone HMB-45, dilution 1:100, HIER - pH 6/20 min/100 °C, incubation for 30 min). Ten high power fields (HPF) equally distributed within the tumor were acquired from each case using a Leica DM RA microscope equipped with a Leica DFC420 C digital camera and processed using the Leica Application Suite version 3.8.0 (Leica Microsystems, Switzerland). Quantitative morphometry was performed using the ImageJ public domain Java image processing program as described in the supporting methods [47 ].

For spheroid formation, adherent FHC-silenced and non-silenced SKOV3 cells were trypsinized and two different cell concentrations per well (1 × 10³/ml and 1 × 10²/ml) were seeded in ultra low attachment 6-well plates (Corning Incorporated, Corning, NY, USA), using sphere medium described above. After six days, the first generation spheroids were dissociated using 500 μl accumax (Millipore, Temecula, CA, USA) and then centrifuged at 200 × g for 5 min. Dissociated cells were resuspended in sphere medium and plated in second generation. The same procedure was performed to obtain all subsequent generations. Spheres were counted and photographed (magnification of 10×) using the Leica DFC420 C and Leica Application Suite Software.

For evaluating cell morphology, cells were cytocentrifuged onto glass slides, fixed in methanol and stained with May Grunwald-Giemsa (Thermo Fisher Scientific) and photographed with 40× magnification with a digital camera Leica DFC420 C and Leica Application Suite Software (v1.9.0, Leica).

Adipogenic differentiation was induced by culture medium consisting of DMEM (PAA) containing 10% HPL, 0.5 mM isobutylmethylxanthine (IBMX; Sigma, St. Louis, MO, USA), 1 µM dexamethasone (Sigma) and 10 µM insuline (Sigma) as described before [40] (link), [41] (link). Osteogenic differentiation medium consists of DMEM-low glucose (PAA) with 2 mM L-glutamine (Sigma), 100 U/mL pen/strep (Lonza), 100 nM dexamethasone (Sigma), 200 µM L-ascorbic acid-2-PO₄ (Sigma), 10 mM β-glycerophosphate (Sigma). For staining of lipid droplets we initially tried the conventional staining method with Oil red but this dye has high affinity for the PVDF-substrates. Therefore, we stained fat droplets with the green fluorescent dye BODIPY (4,4-difluoro-1,2,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene) counter-stained with DAPI (4′,6-Diamidin-2-phenylindol; both Molecular Probes, Eugene, Oregon, USA). Fluorescence microscopy pictures were taken from representative areas. Images acquisition was performed using a Leica DM IL LED microscope (Leica, Wetzlar, Germany) with a 10x dry objective (numerical aperture: 0.3; Leica) and a camera (Leica DFC420C) equipped with Leica application suite 3.3.1 software. For analysis of calcium deposits upon osteogenic differentiation we tested Alizarin Red as described before [42] (link).

Overviews of sectioned lungs were documented either with a DM5000 or DM6000 microscope (Leica Camera, Wetzlar, Germany) equipped with a Leica DFC300FX or Leica DFC350FX digital camera, respectively. For higher magnifications confocal microscopy was performed using a Leica DM IRB with a TCS SP2 AOBS scan head. Organ cultures were photographed with the DM6000 microscope, a Leica M420 microscope with a Fujix digital camera HC-300Z (Fujifilm Holdings, Minato/Tokyo, Japan) or a Leica MZFLIII with a Leica DFC420C digital camera. Images were processed and analyzed in Adobe Photoshop CS5 (Adobe, San Jose, CA, USA).