Bx 51 upright light microscope

TMA scoring was performed by a board-certified, experienced pathologist (A.N.) who was masked to the clinicopathologic characteristics of the tumors. An Olympus BX51 upright light microscope was used to score cores at a magnification of 40×. TAP1 and TAP2 antibody staining of cores containing tumor were assessed using a quick-score method [31 (link)]. Cores were scored on a 0–3 scale on the basis of staining intensity (no staining = 0; light staining = 1; moderate staining = 2; strong staining = 3). The proportion of cells staining was also recorded as a percentage. Percentages were later converted to a score on a 1–6 scale (0%-4% = 1; 5%-20% = 2; 21%-40% = 3; 41%-60% = 4; 61%-80% = 5; 81%-100% = 6). The 2 scores were multiplied to obtain the final score, which could range from 0–18 (Table 1). The maximum quick score from each patient’s triplicate specimens was used for analysis. Cores from normal liver and placenta were used as negative controls.

Live flies were fixed in 4% paraformaldehyde, 2.5% glutaraldehyde as previously described [11 (link)]. Transverse sections of approximate midbrain were obtained by taking 150 500 nm slices from the first appearance of tissue and mounting the slice at the final plane. Light microscopy was performed on 500 nM-thick horizontal sections stained with 1% toluidine blue and 1% borax in water. Bright field microscopy was performed using an Olympus BX-51 upright light microscope outfitted with an Olympus DP-70 camera and software. Both central brain and optic lobe were analyzed for appearance of vacuoles by both number and % area using ImageJ (Bethesda, MD) software. Data were exported to Microsoft Excel for quantification. Vacuolization of brain tissue was not found to be significantly different between brain regions analyzed (optic lobe and central brain) and were subsequently pooled. Vacuolar area was not found to be significantly different between Berlin K RU- and RU+ nor between sws⁵ RU- and RU+ and were similarly pooled for analysis.

DNA fragmentation was evaluated using an ApopTag1 (Qbiogene) kit. In this method, the enzyme terminal deoxynucleotidyl transferase labels the 3-OH ends of DNA generated during apoptosis with biotinylated nucleotides. Immunoperoxidase staining was used to detect these fragments. The apoptosis detection kit enables distinction of apoptosis from necrosis by specifically detecting the DNA cleavage and chromatin condensation associated with apoptosis. Human melanoma cells were cultured on 8-well slides (20,000 cells per slide, Roth Germany) overnight for attachment. Then, the cells were treated with the investigated compounds at concentrations of 6 and 12.5 µM for 24 h. After treatment, the cells were fixed with 4% formalin in PBS for 10 min and permeabilized with 0.2% Triton X-100 in PBS for 5 min at room temperature. TUNEL assay was carried out according the manufacturer’s (Millipore) instructions. Nuclei were stained with hematoxylin. Then samples were mounted by DPX (Thermo Fisher Scientific, Germany) on glass slides. Cells with stained nuclei were investigated by counting 100 cells in 3 randomly selected fields. The analyses were performed by two independent investigators. Samples were evaluated with a BX51 upright light microscope (Olympus, Japan).