Dp30bw digital camera

Giemsa-stained metaphase chromosomes were studied under a Carl Zeiss AxioImager.Z2 microscope, equipped with Metafer Scanning Platform (Metasystems) and a MetaSystems CoolCube digital camera. Images were processed for karyotype reconstruction with Ikaros karyotyping software (Metasystems). For C-banding, FISH and CGH methods, images from at least 20 metaphase chromosomes were analyzed using a Provis AX70 (Olympus) fluorescence microscope, equipped with a DP30BW digital camera (Olympus). All images were acquired in black and white, and later superimposed with colours in DP Manager imaging software (Olympus).

A Zeiss LSM 780 LSCM confocal microscope (Carl Zeiss AG) with a 25× glycerine immersion objective (NA 0.8) was used to image the samples. A resolution of 0.33 µm×0.33 µm×0.5 µm was used for all the images. After imaging, the 3D stacks were deconvolved with Huygens Essentials software (Scientific Volume Imaging, Hilversum, Netherlands). The deconvolved colour channels were merged using ImageJ (U.S. National Institutes of Health). An Olympus IX51 inverted fluorescence microscope and an Olympus DP30BW digital camera (Olympus Corporation) with a 10× objective (NA 0.30) without immersion medium were used to image the samples in 2D format. ImageJ was used to visualize the 2D images.

Images were captured using a Provis AX70 (Olympus, Tokyo, Japan) fluorescence microscope equipped with a DP30BW digital camera (Olympus Tokyo, Japan). The karyotype was arranged using Ikaros karyotyping software (Metasystems, Altlussheim, Germany). DP manager imaging software (Olympus, Tokyo, Japan) was used to capture greyscale images and to superimpose the source images with colours to visualize the results of the FISH.

Cells were seeded (5×10⁴) in 1% agar-coated 96-well plates and cultured for 48 h in a humidified atmosphere containing 5% CO₂ at 37°C. Intact tumor spheroids were carefully transferred to a 96-well plate and cultured in complete media for 24 h. Spheroids and migrated cells were fixed with 100% methanol, stained with 0.05% crystal violet, and observed using a normal light microscope (20×) and Olympus DP-30BW digital camera. Eight replicates were included per group, and experiments were repeated three times.

The photos of metaphases were captured by the Provis AX70 (Olympus, Tokyo, Japan) fluorescence microscope equipped with a DP30BW digital camera (Olympus). The photos from FISH experiments were further processed using the DP manager imaging software (Olympus, Tokyo, Japan). Karyograms were constructed from Giemsa-stained preparations in the Ikaros karyotyping software (Metasystems, Altlussheim, Germany).

Optimal cutting temperature compound-embedded 6 µm-thick frozen tumor sections were processed for immunofluorescence analysis as per the standard protocol. Sections were blocked with 2% bovine serum albumin prepared in PBS and incubated with an anti-CD31 antibody (1:100 dilution; Santa Cruz Biotechnology) overnight at 4°C, followed by an Alexa546-conjugated goat anti-mouse secondary antibody (1:200 dilution; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI; Sigma) to visualize cell nuclei. Immunofluorescence images were acquired under an Olympus DP30BW digital camera. CD31-positive cells present in tumor sections were quantified using MetaMorph software, version 7.1.6.0 (Molecular Devices, Sunnyvale, CA, USA).
HEK293E cells were plated on serum-coated coverslips and transfected with Sp1-specific siRNA and the ZEB2 expression vector for 48 h. Cells were fixed for 5 min in 10% formalin and permeabilized in 0.3% Triton X-100 for 3 min. Cells were incubated with an anti-ZEB2 antibody (Active Motif) followed by a Alexa546-conjugated secondary antibody and then with an anti-Sp1 antibody (Santa Cruz Biotechnology) followed by a FITC-conjugated secondary antibody. Cells were counterstained with DAPI. Mounted samples were visualized with a confocal microscope (LSM 510 META; Carl Zeiss, Jena, Germany).