Coolpix 990

Whole-mount in situ hybridization was performed using digoxigenin-labeled antisense RNA probes followed by detection with anti-digoxigenin alkaline phosphatase-conjugated antibody as described previously [27] (link). The pGEM-T-gnrh3 plasmid was linearized with NcoI before being used as a template for in vitro transcription with SP6 RNA polymerase. After in situ hybridization, staining and mounting, images were captured with a digital camera (Coolpix 990, Nikon).
For double staining, after in situ hybridization, digoxigenin-labeled gnrh3 antisense RNA probe was first reacted with mouse anti-Digoxin conjugated DyLight 488 (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) followed by immunochemical reaction with rabbit anti-GFP antibody (sc-8334, Santa Cruz, CA, USA) and detection with Alexa Fluor 647-conjugated to donkey anti-rabbit IgG antibody (Invitrogen Corporation, Carlsbad, CA, USA). The signals were observed using a Leica TCS-SP5-MP-SMD confocal system (Leica Microsystems Wetzlar, Wetzlar, Germany).

Film structure was observed using the light microscope (Vision Engineering, Emmering, Germany) with coupled camera (Nikon Coolpix 990, Japan). The uncoated plum skin, the skin of plums immediately after dipping, and the skin of plums coated and dried were focused up to 400x, and magnifications ranging from 10× to 40× were selected. No special preparation was necessary for microscopic observation.
Film microstructure was also observed using a polarizing microscope BX51 (Olympus, Japan). Therefore, small sections of plum skin (approximately 1.0 × 0.5 cm) were cut off with a sharp razor blade. One untreated skin sample was observed directly and two other samples were coated by dipping the skin into a solution of starch film-forming solution. Coated plum skin was observed directly and after air drying. For light microscopic analysis, the samples were placed on microscopic slides and illuminated from the top side with an external light source (100 Watt gooseneck lamp). For epifluorescence images, an Olympus U-MBFL3 reflected light brightfield mirror cube equipped with a built-in neutral density filter was used. Excitation was performed with an Exfo X-Cite 120Q lamp. Images were taken at magnifications of 1000×, 2000× and 4000×.

Muscle sections were viewed under a light microscope (Eclipse E400, Nikon, Badhoevedorp, Netherlands) connected to a digital camera (Coolpix 990, Nikon). Photographs were taken at different magnifications (from x40 to x400) for (i) fiber type, (ii) cytochrome c oxidase (COx) activity, and (iii) capillary density (CD). For these analyses, an average of eight fields was examined in each section, allowing for inspection of 150 fibers per individual sample. All photographs were analyzed using Scion image analysis free software (Scion Image Software, Scion Corporation, Frederick, Maryland, USA).

NIH3T3/EGFRvIII (1 × 10⁵) cells or NIH3T3/EGFRwt (1 × 10⁶) cells were suspended in 100 μL of 62.5 % v/v Matrigel (BD Biosciences) in DMEM and transplanted subcutaneously into female Balb/c nude mice at 7–8 weeks of age. Either Ertredin derivatives or gefitinib were dissolved in a vehicle consisting of saline, 10 % w/w DMSO, and 1 % w/w Tween80. After cell transplantation, 30 mg/kg Ertredin or I-Ertredin was injected intraperitoneally once daily from day 1–17. Gefitinib at 100 or 200 mg/kg was orally administrated to mice transplanted with these cells as a positive control.
Five to 6 mice were included in the vehicle-injected group, 4–5 in the Ertredin-or I-Ertredin-injected group, and 3–5 in the gefitinib-injected group. We measured the long and short diameters of the tumors every 2 or 3 days, and calculated tumor volume with the following equation: tumor volume = (long diameter) × (short diameter)² / 2. On day 17 or 18, the tumors were extirpated, weighed, and photographed using a Nikon COOLPIX 990. The images were processed by Preview (MacOS). For TUNEL staining, parts of the tumors were fixed using 4 % paraformaldehyde phosphate buffer solution (Wako), followed by paraffin embedding.

Cells (1 × 10⁵/well/6-well plate) were stimulated, or not, as described above, and stained for lipid droplets (LD) by using Oil Red O (ORO), since ORO selectively stains and detects neutral lipids. Briefly, cells were fixed in 3.7% formaldehyde (10 min), rinsed with PBS and incubated with 100% propylene glycol (5 min). Next, stained with ORO (Sigma) (10 min) pre heated at 60 °C, followed by 5 min incubation with 85% propylene glycol, then rinsed with PBS and incubated with DAPI (Molecular Probes-Invitrogen) (5 min). Photos of the stained cells were taken in a light microscope (Nikon, Tokyo, Japan) connected to a digital camera (Coolpix 990; Nikon). The number of LD per cell was determined by the software ImageQuant TL (GE Healthcare), using the option of colony counting.

Immunohistochemistry (IHC) was performed on 3 μm paraffin sections of 19 ESCC cases and their respective normal surrounding mucosa. For HMGA1 and HMGA2 antigen retrieval, sections were incubated in water bath while submerged in a target buffer solution (DAKO), pH 9.0, for 40 minutes at 98°C. Sections were then incubated with the primaries monoclonal antibodies against HMGA1 (Abcam AB129153, working dilution 1:500) and HMGA2 (Abcam, AB52039, working dilution 1:50), during 12 hours, at least. FFPE anaplastic thyroid carcinoma samples served as positive controls of HMGA1 and HMGA2 staining. As negative control, the primary antibody was replaced by the diluent solution. The detection system used was the NovoLink™ Max Polymer Detection System (Leica Biosystems), following the protocol described by the manufacturer, using diaminobenzidine as substrate - DAKO. Sections were counterstained with Harris’ hematoxylin. The staining score evaluation was performed by three independent pathologists using a light microscope (Nikon, Tokyo, Japan) connected to a digital camera (Coolpix 990; Nikon). For both proteins, scored cases were considered 1+ when positive staining was present in up to 25% of tumor region; 2+ when staining was present between 26% and 50%, 3+ when staining was present between 51% and 75% and 4+ when staining was present between 76% and 100% of tumor region.