Formvar coated copper grids

For transmission electron microscopy (TEM) analysis, 1 mm-thick coronal brain sections were collected at approximately -2.7 mm from the bregma. From these sections, the corpus callosum was dissected, and the portion from the right hemisphere was used for TEM. These dissected samples were immersed in a fixative solution containing 2.5% glutaraldehyde and 4% PFA in 0.1 M sodium cacodylate buffer (pH 7.2). The samples were placed in a vacuum oven for 1 hour, then rotated for 3 hours at room temperature. The fixative solution was subsequently replaced with fresh fixative, and samples were left rotating overnight at 4°C. After washing, the samples were post-fixed overnight in 1% OsO₄ with 1.5% K₃Fe(CN)₆ in 0.1 M sodium cacodylate buffer at 4°C. The samples were rinsed in distilled water and left in 1% uranyl acetate for 1 hour for bulk staining. Samples were then dehydrated through a graded ethanol series, followed by embedding in Spurr’s resin. Ultrathin sections were cut perpendicular to the corpus callosum using an ultramicrotome (Leica EM UC6) and were transferred to 0.7% formvar-coated copper grids (Aurion). Grids were viewed with a JEM-1400plus transmission electron microscope (JEOL, Tokyo, Japan) operating at 80 kV. ImageJ was used to calculate the g-ratio (the ratio of the inner axonal diameter to the total outer diameter) using 8-10 images per animal. MyelTracer software (67 (link)) was used to count the number of myelinated axons. The researchers were blinded during sample processing.

L-PRF samples were fixed with 2% glutaraldehyde (Laborimpex, Brussels, Belgium) in 0.05 M cacodylate buffer (pH 7.3; Aurion, Wageningen, The Netherlands) at 4 °C for ultrastructural analysis.
For Transmission Electron Microscopy (TEM), post-fixation was carried out with 2% osmiumtetroxide (Aurion) for 1 h at 4 °C. Dehydration of the samples was performed in ascending concentrations of acetone. Afterward, the dehydrated samples were impregnated overnight in a 1:1 mixture of acetone and araldite epoxy resin. Next, the samples were embedded in Araldite epoxy resin at 60 °C and were cut in slices of 70 nm with a Leica EM UC6 microtome. The slices were transferred to 0.7% formvar-coated copper grids (Aurion). Afterward, the samples were contrasted with 0.5% uranyl acetate and lead citrate (Laurylab, Saint-Fons Cedex, France) using a Leica EM AC20. Analysis was performed with a Philips EM208 S electron microscope (Philips, Eindhoven, The Netherlands) equipped with a Morada Soft Imaging System camera with iTEM-FEI 4.07 software (SIS, Olympus, Tokyo, Japan).
For Scanning Electron Microscopy (SEM), L-PRF clots were transferred through a gradual ethanol gradient (25, 50, 70, 90%, 2 × 100%, for 10 min each), before being critical point dried with carbon dioxide. Subsequently, specimens were mounted on 12 mm aluminum stubs, and sputter coated with a ∼5 nm thick gold/palladium layer (JEOL JFC-1200 Fine Coater, JEOL, Tokyo, Japan). Images were made with a JEOL JSM-5600 LV (JEOL, Tokyo, Japan) under a high vacuum operated at 10–30 kV.