G2 spirit biotwin electron microscope

An aliquot of small EVs resuspended in PBS were fixed with 2% paraformaldehyde (PFA) for TEM. After deposition of PFA-fixed small EVs on Formvar-carbon coated grids (TAAB Laboratories Equipment, Berkshire, UK), grids were contrasted with uranyl acetate for 5 min. Observations were carried out under TECNAI G2 Spirit BioTWIN electron microscope (FEI) at 100 kV.

Exosomes were fixed with 2% paraformaldehyde (PFA) and deposited on Formvar-carbon coated grids (TAAB Laboratories). Samples were washed with PBS and fixed with 1% glutaraldehyde for 5 min. After washing with distilled water, grids were contrasted with uranyl-oxalate pH 7, for 5 min, and transferred to methyl-cellulose-uranyl acetate, for 10 min on ice30 . For immunogold staining, exosomes were adsorbed on Formvar-carbon coated grids, permeabilized with 0.1% saponin, washed and blocked with 0.5% BSA. Exosomes were incubated with rabbit anti-Cx43 (H-150) and mouse anti-CD63 (MX-49.129.5), washed and labelled with secondary antibodies conjugated to gold particles. Grids were contrasted as described above. Observations were carried out using a Tecnai G2 Spirit BioTWIN electron microscope (FEI) at 80 kV.

Optic nerve samples were collected at approximately 1 mm from the optic chiasm and fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2), supplemented with 1 mM calcium chloride for 2 h. Following rinsing in the same buffer, post-fixation was performed using 1% osmium tetroxide for 1 h. Samples were then washed in buffer and dehydrated in a graded ethanol series (30–100%), impregnated and embedded in Epoxy resin (Fluka Analytical, Sigma-Aldrich, St. Louis, MO, USA). For the evaluation of whole nerve and individual axons, ultrathin sections (70 nm) were mounted on copper grids (300 mesh) and stained with 2% uranyl acetate (15 min) and 0.2% lead citrate (10 min). Observations were carried out using a Tecnai G2 Spirit BioTWIN electron microscope (FEI) at 100 kV.
The electron microscopy images were graded in a masked-fashion by an independent observer for optic nerve axonal damage (Grade 1 = 0 to 25%, Grade 2 = 25 to 75%, Grade 3 = 75 to 100% of axons with myelin disarrangements). A second independent observer was asked to grade a subset of the images and κ-statistic was computed to measure inter-observer agreement. A strong inter-observer agreement was found (κ = 0.607, p < 0.001). The results were presented as the frequency distribution of each grade attributed to each animal.