Digital sight ds fi1

The wound healing assay [6 (link),7 (link)] was used to test SH-SY5Y cell migration. Cells were plated in 6-well plate at 5 × 10⁵ cells/well density in complete medium. Once cells reached confluence, a longitudinal scratch was performed through the cell monolayer using a 200 μL sterile plastic tip. Plates were then washed three times with PBS to remove non-adherent cells. Fresh starvation medium containing POE, NP-POE, PM-POE, or empty nanoformulations at appropriate dilutions was added. Cell-free area was observed under phase contrast microscopy and images were captured at 0, 5, 7, and 24 h after wounding using a Nikon TS-100 microscope equipped with a digital acquisition system (Nikon Digital Sight DS Fi-1, Nikon, Minato-ku, Tokyo, Japan). Marked edges along each wound were used to measure cell migration considering the horizontal distance between the initial scratch and the scratch following migration.

Tumors retrieved from mice were fixed in 10% formalin (VWR Chemicals, 90240) and analyzed. Samples were then embedded in paraffin, cut into 4 μm sections. Histological examination was performed using a light microscope – Nikon Eclipse 50i, and images obtained using a Nikon-Digital Sight DS-Fi1 camera. Immunohistochemistry studies were performed on a representative block of the lesion, performed on Ventana Marker Platform Bench Mark ULTRA IHC/ISH, with an indirect multimeric detection system, biotin free, peroxidase conjugated, with anti-KI-67 antibody (Santa Cruz Biotechnology Cat# sc-23900, RRID:AB_627859).
The Ki-67 proliferative index was counted accordingly to the manual counting of camera-captured/printed image method defined for neuroendocrine tumors (Reid et al., 2015 (link)): each slide was manually scanned under a × 10 objective, for selection of the area with greatest Ki67 positivity (hot spot), with posterior photograph and print. The color image was printed on plain white paper and Ki67-negative and -positive tumor cells were then visualized and immediately marked/crossed off once counted. Light brown/pale staining nuclei were ignored for the purpose of counting.

HepG2 cells were seeded at a density of 6 × 10⁴ in 24-well plates in NG growth medium and incubated overnight. Next, cells were treated in both NG and HG conditions with EXT in the range of 0.04 to 0.30 µg/mL (w/v) or ME-EXT, at the corresponding concentrations of EXT. Cells untreated or treated with corresponding dilutions of the empty ME were used as controls. After 24 h incubation, cells were fixed in 2% (v/v) paraformaldehyde for 10 min. After two PBS washes, wells were left to dry at 37 °C for a few minutes. At this point, a 60% (v/v) ORO solution in bi-distilled water was added at 200 µL/well and incubated at 37 °C, with stirring for 30 min. Subsequently, repeated washes in bi-distilled water were carried out to remove the excess dye. Images of intracellular lipid staining were captured using a Nikon TS-100 microscope equipped with a digital acquisition system (Nikon Digital Sight DS Fi-1; Nikon, Minato-ku, Tokyo, Japan). The staining intensity was measured by solubilizing the dye with isopropanol (200 µL/well). The absorption values were measured with an iMARK microplate reader (Bio-Rad Laboratories, Hercules, CA, USA) at a wavelength of 490 nm [75 (link)]. Cell viability values were used to normalize the ORO assay data. Values are reported in terms of the percentage with respect to untreated control cells.

Aortas were pinned to black wax plates and stained with Oil Red O stock solution (1.8% v/v). Stained aortas were immersed in phosphate buffered saline (PBS) and photographed using a Nikon Digital Camera (Nikon Digital Sight DS-Fi1) mounted on Nikon Dissecting Microscope (SMZ 1000, Micron-Optics, Cedar Knolls, NJ). Image analysis was performed by NIS-Elements: Basic Research Version 3.0 (Micron-Optics, Cedar Knolls, NJ). Oil Red O stained lesion areas were measured and expressed as a percentage of total aorta area as described previously. To characterize plaque composition, histological analysis of lesions in the aortic root was performed via the Paigen method. Serial sections were stained with trichrome for lesion area measurements. Macrophage content was evaluated by CD68-positive staining via immunohistochemistry. A rat anti-mouse CD68 antibody (Cat. #: MCA1957; AbD Serotec, Raleigh, NC) was applied to paraffin-embedded and sectioned samples. Samples were processed as described [14] (link).

For immunohistochemistry study, frozen TA muscle sections or 3.7% PFA fixed single myofibers or cultured myogenic cells were blocked in 1% bovine serum albumin in PBS for 1 h and incubated with anti-Pax7 (1:10, Developmental Studies Hybridoma Bank, DSHB, University of Iowa, Iowa City, IA), anti-MyoD (1:200, Santa Cruz Biotechnology), anti-E-MyHC (1:200, DSHB, University of Iowa, Iowa City, IA), anti-MF20 (1:250, DSHB, University of Iowa, Iowa City, IA), anti-laminin (1:100, Sigma Chemical Company) in blocking solution at 4 °C overnight under humidified conditions. The sections were washed briefly with PBS before incubating with Alexa Fluor 488- or 546-conjugated secondary antibody (1:3,000, Invitrogen) for 1 h at room temperature and then washed three times for 5 min with PBS. The slides were mounted using fluorescence medium (Vector Laboratories) and visualized at room temperature on Nikon Eclipse TE 2000-U microscope (Nikon), a digital camera (Nikon Digital Sight DS-Fi1), and Nikon NIS Elements BR 3.00 software (Nikon). Image levels were equally adjusted using Adobe Photoshop CS6 software (Adobe).

For immunohistochemistry studies, frozen TA or plantaris muscle sections were fixed in acetone or 4% PFA in PBS, blocked in 2% BSA in PBS for 1 h, and incubated with mouse anti-Pax7, mouse anti-eMyHC, and rabbit anti-laminin, in blocking solution at 4°C overnight under humidified conditions. The sections were washed briefly with PBS before incubation with goat anti-mouse Alexa Fluor 594 and goat anti-rabbit Alexa Fluor 488 secondary antibodies for 1 h at room temperature and then washed three times for 15 min with PBS. Nuclei were counterstained with DAPI. The slides were mounted using the fluorescence medium (Vector Laboratories) and visualized at room temperature on a Nikon Eclipse TE2000-U microscope (Nikon), a digital camera (Nikon Digital Sight DS-Fi1), and NIS-Elements BR 3.00 software (Nikon). Image levels were equally adjusted using Photoshop CS6 software (Adobe).

For immunohistochemistry studies, frozen TA or plantaris muscle sections were fixed in acetone or 4% paraformaldehyde (PFA) in PBS, blocked in 2% bovine serum albumin in PBS for 1 h and incubated with rabbit anti-dystrophin, mouse anti-Pax7, mouse anti-eMyHC, rabbit anti-laminin, in blocking solution at 4 °C overnight under humidified conditions. The sections were washed briefly with PBS before incubation with goat anti-rabbit Alexa Fluor 468, goat anti-mouse Alexa Fluor 594, goat anti-rabbit Alexa Fluor 488 or goat anti-mouse Alexa Fluor 488 secondary antibody for 1 h at room temperature and then washed three times for 15 min with PBS. Nuclei were counterstained with DAPI. The slides were mounted using fluorescence medium (Vector Laboratories) and visualized at room temperature on Nikon Eclipse TE 2000-U microscope (Nikon), a digital camera (Nikon Digital Sight DS-Fi1), and NIS Elements BR 3.00 software (Nikon). Image levels were equally adjusted using Photoshop CS6 software (Adobe).