Zen 2.6 blue edition software

The larvae were washed and subsequently stained with Nile Red (Sigma, Jerusalem, Israel), which specifically stained neutral lipids, such as triglycerides [64 (link)]. The larvae were exposed to Nile Red (500 ng/mL) for 0.5 h and subsequently euthanized. Using a 1% low melting point agarose or a glycerol-based Mounting Medium (ibidi Mounting Medium, ibidi, Grafelfing, Germany), each larva was positioned on its right side (location of adipocyte deposit) on a coverslip assembly, based on a published diagram [65 ] (wiki.zfin.org/display/prot/Viewing+Chambers, last accessed on 2 July 2023). The degree of obesity was assessed by the number of Nile Red-stained lipid droplets (assumed to be in adipocytes) and their area, as determined by confocal microscopy (Zeiss LSM 700 laser scanning confocal microscope, using an X5 objective, laser settings of 488 nm for excitation and ≥539 nm for emission and ZEN 2.6 (blue edition) software (Zeiss)). Conditions, including the laser power, pinhole, master gain, and digital offset were kept constant for each experiment. The layer counted was the one with the most Nile Red-stained lipid droplets in the abdominal area. Lipid droplets smaller than 100 µm² were excluded, as were those appearing in a row (since they could be fat droplets in lymph vessels and not adipocytes).

All slides were scanned at 20x resolution using an Axioscan Z1 slide scanner (RRID:SCR_020927, Zeiss, Jena, Germany). Zen 2.6 Blue Edition® software (RRID:SCR_013672, Zeiss, Jena, Germany) was used to globally adjust the copies of original photomicrographs for white and black balance. Photomicrographs were additionally cropped, rotated and resampled to allow alignment with other stainings. For image analysis three 500 × 500 pixel regions in each patient sample were selected based on number of nuclei (100 ± 20), nestin positivity and adjacency to denser tumor tissue. Then TMs were measured manually in these regions using Fiji 2.0.0. There were 20–84 TMs measured per image with a total of n = 898.

Fluorescence and bright field images were acquired using a Carl Zeiss Axio Imager Z1 fluorescence microscope with ZEN 2.6 blue edition software (Carl Zeiss MicroImaging, Inc.) equipped with ApoTome, AxioCam MRm, and AxioCam HRc cameras. Multi-channel image acquisition was used to create images with multiple channels. Tiling mode and post-acquisition stitching were used to capture and analyze large areas. Images were repeatedly captured at the same focal planes with the same exposure time. Images were captured at 20 × , 40 × , 63 × , and 100 × objectives for different purposes.

A431 cells were fixed either with 4% paraformaldehyde followed by 0.1% Triton X-100 to allow permeabilization or with cold methanol at −20 °C. Cells were then incubated with the following primary antibodies: anti-vimentin rabbit polyclonal antibody (1:400) (Abcam, Cambridge, UK), anti-fibronectin mouse monoclonal antibody (1:400) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-E-cadherin monoclonal antibody (1:200) (Dako, Santa Clara, CA, USA). The primary antibodies were visualized using goat anti-rabbit Alexa Fluor 546 conjugate, goat anti-mouse Alexa Fluor 546, and goat anti-mouse Alexa Fluor 488 conjugate antibodies (1:800) (ThermoFisher Scientific, Monza, Italy). Coverslips were mounted using ProLong Gold antifade reagent with DAPI (Invitrogen, Waltham, MA, USA). The fluorescence signal was evaluated by recording stained images, using a CCD camera (Zeiss, Oberkochen, Germany). For vimentin and fibronectin stainings, the number of positive cells was measured by counting at least 150 cells randomly taken from 10 different microscopic fields, and results are expressed as fold change with respect to control. Quantitative analysis of E-cadherin fluorescence intensity was performed using the Zen 2.6 (blue edition) software (Zeiss, Oberkochen, Germany) and the results are expressed as mean fluorescence intensity/cell ± SD.

Regular light microscopy was performed using the Olympus CX41 microscope (Olympus Life Sciences, Tokyo, Japan). Colony images were photographed with a SZX16 stereoscope (Olympus Life Sciences, Tokyo, Japan). Images were acquired with an AxioCam MRm camera and processed with the software Zen pro.
Fluorescent microscopy was performed using a Zeiss Imager M2 microscope (Zeiss, Oberkochen, Germany). Images were acquired with an AxioCam MRm camera and processed with the software Zen pro. The fluorescence intensity of 50 individual cells from each strain imaged at ×63 magnification was quantified using ZEN 2.6 Blue edition software (Zeiss, Oberkochen, Germany). Fluorescence intensity was quantified using the Zen ‘Histo definition’ quantification software application. Each cell and background were selected using the circular selection tool and the average fluorescence intensity within that circle was recorded. The fluorescence intensity of the background around each cell was measured and served as a blank. The fluorescence intensity of each cell was normalized by subtracting the fluorescence intensity of the cell’s associated blank.