Axioimager z2 fluorescence microscope

For MET amplification detection by FISH, KREATECH Poseidon™ Repeat Free™ C-MET (7q31) and SE7 probe (KREATECH diagnostics, Amsterdam, Holland) was used. Results were analysed using Zeiss AxioImager Z2 Fluorescence microscope (Carl Zeiss Microscopy, Madrid, Spain). Reporting of MET FISH test results has no standardized procedure as MET FISH is not used as a diagnostic tool. In our study, the reporting was done by using 10 % as a cut-off similar to ALK amplification [19 (link)]. If 10 % or more of the cells show a ratio of R: G >2, then the sample is considered MET positive. All samples with more than 5 % MET positivity were re-scored using the PathVysion scoring system, similar to the approved HER2 amplification scoring system in breast cancer. The PathVysion scoring system was done by determining the ratio between the red signals and the green signals (adding the total number of reds in the 20 cells (R) and the total number of greens in 20 cells (G), then dividing R/G and getting a ratio). If the ratio is <2, the sample does not show MET amplification and if the ratio is ≥2, then the sample shows MET amplification (as per PathVysion HER-2 DNA Probe Kit, Abbott laboratories, Illinois, USA).

The localization of SULT1E1 and STS proteins was examined in frozen liver sections. Briefly, frozen sections (6 μm) were fixed with acetone at −20 °C for 5 min. Then, the endogenous activity of peroxidase was blocked by treating the sections with 3% hydrogen peroxide for 10 min followed by exposure to an Avidin/Biotin blocking kit (Vector Laboratories, Burlingame, CA, USA, #SP-2001). The nonspecific-binding sites were blocked by incubating with 10% of normal horse serum in PBS for 30 min at RT. Following this step the sections were incubated with the primary antibodies: anti-SULT1E1 (Sigma, #028728, diluted 1:250) and anti-STS (Abcam, #62219, diluted 1:100) for 24 h at 4 °C. Then, the tissue samples were rinsed and incubated with a biotinylated anti-mouse/anti-rabbit IgG (#BA-1400, Vector Laboratories, Burlingame, CA, USA ) secondary antibodies for 60 min at RT. The reactions were visualized using ABC Vectastain and DAB kits (Dako, Agilent Technologies, Hovedstaden, Denmark). Then, the sections were counterstained with Mayer’s hematoxylin, dehydrated, and mounted. The negative control, in which the primary antibodies were omitted, was included in the study and uniformly demonstrated no reaction. Images were acquired with a ZEISS Axio Imager Z2 fluorescence microscope (Zeiss, Oberkochen, Germany).

To analyze the progression of sporulation, 1 × 10⁷ cells were collected by centrifugation, washed in water and fixed in 70% ethanol for 45 min. Cells were then stained with DAPI (4′,6-diamidino-2-phenylindole; excitation at 359 nm and emission at 457 nm) at a final concentration of 1 mg L⁻¹ and washed in water two times prior to microscopy analysis. For the analysis of spore-wall formation, cells were collected by centrifugation, washed in water and stained with the chitin/chitosan-binding dye calcofluor white (CFW; excitation at 365 nm and emission at 435 nm) at a final concentration of 25 µM. To determine the percentage of dead cells after exposure to CdS QDs, yeast cells grown in sporulation medium were collected by centrifugation, washed in water, and stained with 100 mg L⁻¹ of propidium iodide (PI; excitation at 536 nm and emission at 617 nm), a membrane-impermeant DNA dye excluded from viable cells, but internalized by dead cells. Zeiss Axio Imager.Z2 fluorescence microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) was used for image acquisitions. For each condition, 150 cells in three independent experiments were analyzed.

Coverslips with primary neurons were dipped in PBS with 4% sucrose and subsequently fixed in 4% paraformaldehyde with 4% sucrose in PBS for 10 min. After washing, they were incubated in a blocking solution (0.3% TritonX-100 and 5% NGS in PBS) for 30 min. Primary antibody staining was performed on a shaker at RT for 1 h. Neurons were washed and incubated with the secondary fluorescent antibody at RT for 1 h in the dark. Both antibody solutions contained 5% NGS to prevent unspecific binding. Finally, after washing again in PBS, the coverslips were mounted on slides with Dako fluorescent mounting medium. Images were acquired with a x63 oil immersion objective in a VisiScope cell analyzer/Zeiss Axio Imager.Z2 fluorescence microscope (Zeiss, Oberkochen, Germany) equipped with the VisiView software (version 4.0.0.16; Visitron Systems, Puchheim, Germany), and they were analyzed with the ImageJ software (NIH Image, version 2.14.0/1.54f, RRID: SCR_003073).

The localization and level of PDC-E2 and p-STAT3 were examined in frozen liver sections (6 µm) using immunohistochemistry. Briefly, frozen sections were fixed with acetone at -20 °C for 5 min. Then, they were incubated with 3% H₂O₂ followed by exposure to the Avidin/Biotin blocking kit (Vector Laboratories, #SP-2001). After blocking with 10% NHS (Normal Horse Serum) for 30 min, the sections were probed with primary antibodies: anti-PDC-E2 (Santa Cruz, #271534, diluted 1:250) and p-STAT3 (Y705; Cell Signaling, #4113, diluted 1:100) for 24 h at 4 °C. Next, biotinylated anti-mouse/anti-rabbit IgGs (#BA-1400, Vector Laboratories) were used as secondary antibodies. The reactions were visualized using the ABC Vectastain and DAB kit (Dako, Agilent Technologies, Denmark). Additional counterstaining with Mayer’s Hematoxylin staining (DAKO) was also performed. The negative control, in which the primary antibodies were omitted, was included in the study and uniformly demonstrated no reaction. To confirm the membrane localization of PDC-E2, an immunofluorescent assay was performed as described previously^{24 (link)}. Images were acquired with a ZEISS Axio Imager Z2 fluorescence microscope.