C11440 digital camera

Immunohistochemistry and immunofluorescence were performed as previously described (Genovese et al., 2017 (link)). Briefly, tumor samples were fixed in 4% formaldehyde (Fisher Scientific) for 24 hr at room temperature, moved in 70% ethanol (Fisher Scientific) for 48 hr, and embedded in paraffin (Leica ASP300S). After cutting (Leica RM2235), baking and de-paraffinization, slides were treated with Citrate Buffer solution (Electron Microscopy Sciences) according to manufacturer’s instructions. For immunohistochemistry staining, endogenous peroxidases were inactivated in a solution of 3% hydrogen peroxide (Sigma-Aldrich) for 20 min. Non-specific signals were blocked for 1 hr using 10% FBS and 5% BSA (Sigma-Aldrich). Tumor samples were stained with primary antibodies for 12 hr at 4°C. HRP-conjugated secondary antibodies (ImmPress, Vector Lab) and Nova RED peroxidase substrate (Vector Lab) were used for detection. Images were captured using a Nikon EclipseTi microscope and a Nikon DS-Fi1 digital camera. For immunofluorescence studies, Alexa488 and 555 conjugated secondary antibodies (Molecular Probes) were used. Images were acquired with a Hamamatsu C11440 digital camera, on a wide-field Nikon EclipseTi microscope.

Cells (5000 cells/well) were plated on glass coverslips in 24-wells plate for indicated time. Then, cells were washed three times with 1X PBS and fixed using 4.0% formaldehyde for 20 minutes at room temperature. Fixed cells were washed with PBS 1X and permeabilized by using 0.2% Triton X-100 in PBS 1X for 10 minutes. After permeabilization, cells were washed 3 times with PBS 1X and then blocked using 5% goat serum in PBS 1X for 60 minutes at room temperature. Cells were washed three times with PBS 1X and then were incubated with primary antibodies overnight at 4 degrees Celsius. Then, cells were washed three times with 1X PBS and incubated with both an anti-mouse and an-anti rabbit secondary antibody (Alexa Flour 488, Alexa Flour 555-Thermo Fisher Scientific) diluted 1:400 in antibody dilution buffer for 120’ at room temperature in the dark. After three washes with PBS 1X, cells were stained with DAPI (Invitrogen) for 10 minutes. Then cells were washed with PBS 1X and coverslips where mounted on the microscopy slides with antifade mounting medium (VECTASHIELD). After 24 hours fixed images were captured with a Hamamatsu C11440 digital camera, using a wide-field Nikon Eclipse Ni microscope.

Tumor samples were fixed in 4% formaldehyde for 2 to 4 hours on ice, moved in 70% ethanol for 12 hours, and then embedded in paraffin (Leica ASP300S). After cutting (Leica RM2235), baking and deparaffinization, slides were treated with Citra-Plus Solution (BioGenex) according to specifications. For IHC staining, endogenous peroxidases were inactivated by 3% hydrogen peroxide. Non-specific signals were blocked using 3% BSA, 10% goat serum and 0.1% triton. Tumor samples were stained with primary antibodies. For BrdU detection, samples were digested on slides for 1 hour at 37° C with DNAse I (300 µg/ml) before staining. For IHC, ImmPress and ImmPress-AP (Vector Lab) were used as secondary antibodies and Nova RED, Vector BLUE and DAB were used for detection (Vector Lab). Images were captured with a Nikon DS-Fi1 digital camera using a wide-field Nikon EclipseCi microscope. For immunofluorescence, secondary antibodies conjugated with Alexa488 and 555 (Molecular Probes) were used. Images were captured with a Hamamatsu C11440 digital camera, using a wide-field Nikon EclipseNi microscope. LipidTox, Lysotracker, MitoTracker, CellRox and Hoechst 33342 (Molecular Probes) were used on live spheres and cells at the concentrations suggested by manufacturer's protocols and images were acquired using a Nikon high-speed multiphoton confocal microscope A1 R MP.