Axio vert a1 microscope

Cells were cultured in a 6-well plate at 40% confluence and were transfected with FugeneHD (Promega, USA). The fluorescent signal for TFEB-GFP was observed using an Axio Vert.A1 Zeiss microscope with an AxioCam. Images were taken of the cells before and after treatment with 100 nM of wortmannin, following the translocation of TFEB-GFP in the same cells over time at 15, 30, 45 min, and 1 h intervals. Regional intensity measurements of the fluorescent signal in the nucleus and cytoplasm were determined using ImageJ software (version 1.53k), with background signal subtraction for all groups. The ratio of the nucleus/cytoplasm GFP intensity was used to represent the subcellular distribution of TFEB-GFP. The values from each group are represented as means ± SD. Statistical analysis was performed using non-parametric ANOVA with the Kruskal–Wallis test for comparisons between groups.

Primary culture of SVC from paired SAT and EAT of 5 patients were or not treated with ACh for 21 days, twice a week. At the end of treatment, lipid droplets, triglycerides accumulation, were visualized after 15 minutes of incubation with AdipoRed Assay Reagent (Lonza Biologics, SL) at room temperature. Representative images were captured with 20X objective of an inverted Axio Vert. A1 Zeiss Microscope (Carl Zeiss Microscopy GmbH). AdipoRed fluorescence intensity was recorded in a FluoStar Optima fluorimeter (BMG Labtech GmbH) and depicted as fluorescence relative units (RFU). The average of 6 independent wells was calculated per treatment and fat tissue from 5 independent patients.

HIF-2α expression from tumor xenografts was evaluated in paraffin-embedded tumor xenografts belonging to the “Classical in vivo cancer research assay”. The immunohistochemical staining was done in 5 μm deparaffined and hydrated sections. Sections were incubated with an anti-HIF-2α antibody (1:600) (NOVUS, Oxon, UK). Then, samples were washed and incubated with HRP-conjugated secondary mouse anti-human antibody. HRP activity was amplified with DakoEn Vision + Dual Link System-HRP (Dako, Santa Clara, CA, USA) and the visualization was performed with a DAB substrate Kit (Dako, Santa Clara, CA, USA). Samples were counterstained with hematoxylin 0.02% and mounted with DPX mounting medium (Sigma-Aldrich, St. Louis, MO, USA). Images were taken at 10× with an Olympus digital camera (Olympus, Hamburg, Germany) coupled with an Axio Vert A1 Zeiss microscope (Zeiss, Jena, Germany). The Vehicle group was tested as positive control and negative controls were performed in parallel, with omission of the primary antibody incubation step.

After 6 days of culturing, the 3D spheroids were harvested, embedded into HistoPrep tissue embedding media (Thermo Fisher Scientific, San Jose, CA, USA), and frozen at −20 °C. Then, the frozen blocks were cut into 10 μm sections, fixed in a mixture of acetone and methanol (1:1) for 15 min and air-dried at room temperature. To identify the desired ECM proteins, the slides were washed with TBS-T (1x) and kept in blocking solution for 1 h at room temperature. Then, samples were incubated with primary antibodies for 3 h at room temperature. The following primary antibodies were used in the experiments: rabbit monoclonal anti-fibronectin primary antibodies (ab2413, Abcam), rabbit polyclonal anti-type I collagen antibodies (ab34710, Abcam), and rabbit polyclonal anti-laminin antibodies (ab11575, Abcam). After incubation with primary antibodies, the samples were washed with TBS-T and incubated with goat anti-rabbit IgG labeled with Alexa Fluor 488 (ab150077, Abcam) for 1 h at room temperature. Nuclear DNA was stained with DAPI for 10 min before the samples were covered with a cover glass. The images of spheroid sections were obtained using an inverted AxioVert.A1 microscope (Zeiss, Oberkochen, Germany) equipped with a ×20/0.6 objective lens.

SF A549 spheroids were generated using MicroTissues 3D Petri Dish^® micro-molds (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. Briefly, the micro-molds were sterilized with anhydrous ethanol and allowed to dry under UV light for 30 min. Then, they were filled with sterile 2% (w/v) agarose solution, prepared in Milli-Q water. After solidification, the gelled agarose molds were released from the flexible 3D Petri Dish^® micro-molds and transferred to 6-well plates (3 agarose molds per well). To equilibrate the agarose gels, each well was filled with 1 mL of growth medium. After equilibration, the plates were placed in the incubator and incubated overnight.
Before seeding the cells, the medium was removed from both the culture plate and the molds. Then, 1.3 mL of fresh growth medium was added to each well and aliquots of 150 μL of growth medium with 40,000 cells were gently added into the molds. The medium was changed every other day. The size and morphology of the grown spheroids were examined by bright field microscopy using AxioVert.A1 microscope (Zeiss, Oberkochen, Germany) equipped with a Plan 10×/0.25 phase-contrast objective. On day 7, the spheroids were harvested for the following experiments.