Vacuum desk 5

CaOx crystals produced through EC on ITO were characterized by Fourier transform infrared spectroscopy (FTIR/ATR) in an Interspec 200-X^® (Interspectrum OU, Toravere, Estonia) instrument. The morphologies of the CaOx and PCL-ESM samples were observed by optical microscopy (OM) and scanning electron microscopy (SEM) in a Nikon Eclipse E400^® with the morphometric LAZ program (Image Pro-Plus, Media Cybernetics, Melville, NY, USA) and in a JEOL JSM-IT300LV microscope (JEOL USA Inc., Peabody, MA, USA), respectively. For the SEM analysis, PCL-ESM samples were gold-sputter-coated to thickness of 200 nm using a Denton Vacuum Desk V sputtering system in an argon atmosphere to render them electrically conductive. SEM observation was performed using an accelerating voltage of 20 kV. Moreover, the X-ray diffraction (PXRD) (Siemens, Munich, Germany) of PCL-ESM was conducted using a Siemens D-5000X X-ray diffractometer with CuKα radiation (graphite monochromator) and an ENRAF Nonius FR 590 X-ray generator. The crystal structure of the CaOx was determined using CuKα radiation (40 kV), a step scan of 0.2°, and the geometric Bragg–Brentano (θ-θ) scanning mode with an angle (2θ) in the range of 5–60°. The DiffracPlus program was used as the data control software.

The morphological changes observed in B. canis-stimulated human or canine DCs were analyzed by scanning electron microscopy. Briefly, DCs were fixed with 2% glutaraldehyde for 2 h, washed three-times with PBS, dehydrated through immersion in graded ethanol (50, 70, 95 and 100%), and dried at CO₂ critical point. Cells were then mounted on aluminium stubs, sputter-coated with gold layer to a thickness of 200 nm (Denton Vacuum Desk V; Moorestown, NJ, USA), and examined in a scanning electron microscope (Jeol JSMIT300LV; Jeol Ltd, Tokyo, Japan) at an accelerating voltage of 20 kV.

The diameter and surface morphology of bovine, porcine, and fish gelatin nanofibers were examined using a scanning electron microscope (SEM) (JEOL, JSM-6610, Japan) operating at 10 kV. Before imaging, the samples were coated with a 1 nm layer of gold (Denton Vacuum, Desk V, United States)²⁸ . The average diameter of the nanofibers was determined by randomly measuring the diameter of 50 individual nanofibers in a unique random sample.

The EDX technique allows analyzing the samples qualitatively and quantitatively based on the peak emission of X-rays resulting from the interaction between each element of a compound and the electron beam. Then, the chemical components of the melanin were determined by EDX. Melanin extracts were fixed in glutaraldehyde 2% with 0.1 M sodium cacodylate buffer during 2 h and then washed once in the same solution. To dehydrate the extracts, they were exposed to increasing alcohol concentrations (50°, 70°, 95°, and 100° for 5 min). Samples were dried using a critical point dryer (Autosamdri^®-815, Series A) for 45 min through carbon dioxide (CO₂). Once dried, samples were mounted in aluminum sample holders, and then metalized with gold (Denton Vacuum Desk V). Finally, samples were visualized using SEM (JEOL^® Model JSM-IT300LV).