Comprehensive Skin Tissue Analysis Protocol

Samples were fixed by immersion in formaldehyde and treated with different concentrations of alcohol (70, 80, 95, and 100%), followed by xylol and paraffin, and ultimately fixed in paraffin blocks that were subsequently segmented at 5-µm intervals and placed on microscope slides pre-treated with poly-L-lysine. Then, samples were stained with hematoxylin and eosin. The sections were incubated with hematoxylin for 30 s, bathed in distilled water, incubated for 30 s with eosin, cleaned in purified water, and dehydrated. The slides were placed on Entellan^® (Merck Millipore, USA), and the images were digitally taken under bright field microscopy (Zeizz Axiophot, fluorescent-microscope, USA). Picrosirius red staining was applied to examine collagen fibers. For this, sections were immersed in 100% xylene, followed by hydration in 100, 95, and 70% ethanol before being incubated in 0.1% picrosirius red (EasyPath, Brazil) for 1 h, rinsed with water, and incubated in Carazzi's hematoxylin for 4 min, successively, at room temperature. The double refraction patterns were evaluated by microscopy (Nikon^® Corporation, Japan), using C-PL polarized filters. The stained collagen fibers were analyzed using Image-Pro Plus (v4; Media Cybernetics Inc., USA), and were either bright green (type III collagen-like) or red (type I collagen-like). Using the Image J (http://imagej.nih.gov/ij/) and Orientation J plug-in (Biomedical Image Group, EPFL, Switzerland), we measured the quantities, organization, and density of collagen. For these analyses, five representative areas (800×800 pixels) were randomly chosen in each 8-bit image (1920×1080 pixels) (12 (link)). For the Nissl assay, after fixation with formaldehyde, the samples were dehydrated in progressive concentrations of sucrose in PBS solution (20 and 30%), then blocked in Tissue-Tek polymer (Sakura Finetek, USA), and sectioned at 10.0 µm intervals in a cryostat. The slides were immersed in a solution of 2% toluidine blue for 1 min and fixed with Entellan^®. For immunofluorescence analysis, deep-frozen skin tissue samples were cut into transverse sections (10 μm). After blocking with Donkey Serum (D9663; Merck KGaA, Germany) in BSA 0.3% solution, specimens were labeled with anti-BDNF primary antibodies (sc-65513; Santa Cruz Biotechnology, USA). Thereafter, the sections were incubated with Alexa 546 conjugated IgG secondary antibody and TO-PRO™-3 Iodide (642/661) (Thermo Fisher Scientific, USA) for nuclear staining. The images were captured with an upright Zeiss LSM780-NLO Confocal Microscope (Carl Zeiss AG, Germany).