Lsm 780 confocal laser scanning system

For immunocytochemical analysis, the cells cultured in 2D and 3D conditions were seeded on 24-well plates (Nunc, Thermo Fischer Scientific). The seeding number of cells was 7 × 10⁴ per each 13 mm-diameter poly-L-lysine/laminin-coated coverslip. The 2D culture cells were gently washed in PBS (Sigma-Aldrich), fixed with 4% PFA (Sigma-Aldrich) in PBS (Sigma-Aldrich) for 15 min at RT, and again washed in PBS (Sigma-Aldrich). To detect intracellular proteins and to permeabilize cell membranes, 0.2% Triton X-100 (Sigma-Aldrich) was used. Subsequently, a mixture of 10% goat serum (Gibco) and 1% bovine serum albumin (Sigma-Aldrich) was applied for one hour to block nonspecific binding. Next, the samples were washed in PBS (Sigma-Aldrich) and incubated with primary antibodies overnight at 4 °C. For each variant, a negative control was performed. The cells were washed in PBS (Sigma-Aldrich), and the incubation with secondary antibodies was performed in the dark for 2 h. After the cells were washed again in PBS (Sigma- Aldrich), the nuclei were stained using Hoechst 33342 (Sigma-Aldrich) for 15 min. All the samples were analyzed with the LSM 780 confocal laser scanning system and ZEN software (Carl Zeiss). Quantitative analysis was performed as a relation of positive cells to a total cell number (50 cells per repetition). Each variant had 3 repetitions.

For the intracellular detection of NO, cells (cultured DRG neurons) were incubated with 5 μM 4-amino-5-methylamino-2′,7′-difluorescein diacetate (DAF–FM–DA) for 30 min in the absence or presence of 100 μM sodium sulphide. The cells were washed three times and placed in external solution (as used for calcium imaging experiments, see below). The dye was excited with 488 nm laser line on LSM 780 confocal laser scanning system (Carl Zeiss MicroImaging, Jena, Germany) equipped with an Argon laser mounted on an inverted Axio Observer Z1. All experiments were performed in triplicate. Images were processed in ImageJ software where semi-quantitative fluorescence intensity was determined.

S26 cells and cells expressing LMP1 or LMP2A, were grown on glass coverslips (NEST) for 48 hours. Cells were fixed by using 4% paraformaldehyde for 10 min, permeabilized in 0.1% Triton X-100 for 30 min, and blocked in 1% BSA for 1 hour. Then the cells were incubated with anti-Vimentin, anti-ZEB1, or anti-E-cadherin antibodies for 2 hours at room temperature. Fluor Alexa-555 conjugated anti-rabbit IgG and Fluor Alexa-555 conjugated anti-mouse IgG was used as secondary antibody.
For immunofluorescent staining of paraffin-embedded tumors, sections were deparaffinized and rehydrated, followed by heat-mediated antigen retrieval. Slides were permeabilized in 0.1% Triton X-100 for 30 min, blocked in 1% BSA for 1 hour, and then incubated with CD44 PE (1:100 dilution) and CD104 eFluor 660 (1:50 dilution) antibodies at 4°C overnight. Slides were visualized using a Zeiss LSM780 confocal laser scanning system.