Dpc controller software

Immunohistochemistry was performed as previously reported [23 (link)]. The images were obtained using an Olympus BX51 microscope (Olympus, Tokyo, Japan) equipped with various objectives and a DP50 camera. Images were processed using DPC controller software (Olympus).

Tissue arrays were dewaxed and antigens retrieved using high pressure. Endogenous peroxidases were blocked with 3% hydrogen peroxide for 10 min. After immersion in normal goat serum for 30 min, tissues were incubated with the primary antibody at 4 °C overnight, washed with phosphate-buffered saline (PBS), and then incubated with a biotin-conjugated secondary antibody for 30 min at 37 °C. After washing, the sections were incubated with horseradish peroxidase (HRP) complex for 30 min at 37 °C and visualized using diaminobenzidine (DAB). All immunohistochemical images were obtained under an Olympus BX51 microscope equipped with a 20× , a 40× , or 100× objective lens (Olympus, Tokyo, Japan) and a DP 50 camera (Olympus). Images were processed using DPC controller software (Olympus).
Immunohistochemical staining was evaluated by a semiquantitative scoring method. The SLC39A4 staining was scored as follows: no staining (0), light positive staining (1), medium positive staining (2), and strong positive staining (3). The area of positive staining was scored as: <5% (0), 5–25% (1), 26–50% (2), 51–75% (3), and >75% (4). An overall score was calculated by multiplying the intensity and expression scores for each sample. SLC39A4 expression was dichotomized using median staining intensity as the cutoff to define “high” or “low” as above or below the median, respectively.

Cultured cells were fixed with 4% paraformaldehyde, washed twice with PBS, and then blocked with PBS containing 10% normal goat serum. Cells were then stained with anti-E-cadherin, anti-vimentin, or anti-FSP1 polyclonal antibody for 30 min at 37 °C, washed twice with PBS, stained with Cy3 (red) or Alexa Flour 488 (green)-conjugated secondary antibody for 30 min at 37 °C, and then washed twice again before imaging. All immunofluorescence images were obtained with an Olympus BX51 microscope equipped with a 20 × or 40 × objective lens (Olympus) and a DP 50 camera (Olympus). Images were processed using DPC controller software (Olympus).

A JC-10 Mitochondrial Membrane Potential Assay Kit (Solarbio) was used to assess the changes in mitochondrial membrane potential in A549 and H1650 cells after treatment with CC-115 and/or SC79 for 48 h, according to the manufacturer's instructions. The cells were stained with JC-10 working solution for 15 min at 37 °C, washed twice with JC-10 staining buffer, and observed under an Olympus BX51 microscope (Olympus Corporation). The JC-10 aggregate to monomer ratio was analyzed using DPC controller software (Olympus Corporation).

Cells were fixed with 4% paraformaldehyde and then washed with PBS for three times. Normal goat serum was incubated for 40 min, remove the blocking solution, and then add the primary antibody (anti-E-cadherin, anti-Vim, and anti-FSP-1 antibodies) overnight at 4 °C. Following incubation with the primary antibody, cells were washed three times with PBS and stained with the Cy3-conjugated secondary antibody for 30 min at 37 °C. The nuclei were stained with Hoechst 33258. Microscopy was performed after two washes with PBS. All immunofluorescence images were captured on an Olympus BX51 microscope and a DP 50 camera (Olympus). Images were handled by DPC controller software (Olympus).

The immunofluorescence assay was performed according to previous reports [7 (link), 23 (link)]. Immunofluorescence images were obtained using an Olympus BX51 microscope (Olympus, Tokyo, Japan) equipped with various objectives and a DP50 camera. Images were processed using DPC controller software (Olympus).