Ht 7700 elexience

TEM was used to characterize EVs isolated from FF (ffEV) of SF and LF. Transmission electron microscopy analysis was performed on six independent samples per class using the aliquots from each ffEV suspensions described above. A volume of 3 μL of fixed ffEVs was placed on formvar and carbon-coated grid and incubated for 5 min at room temperature. Samples were washed with distilled water trice, then stained with 2% uranyl acetate and air dried at room temperature. The micrographs were obtained using TEM HITACHI HT 7700 Elexience at 80 kV (with a charge-coupled device camera AMT) and JEM 1011 (JEOL, Tokyo, Japan) equipped with a Gatan digital camera driven by Digital Micrograph software (Gatan, Pleasanton, CA, USA) at 100 kV. The images were used to measure EVs size with ImageJ software (NIH, Bethesda, MD, USA). Comparison of microvesicle and exosome size distribution was carried out using Chi2-test by XLSTAT (Addinsoft, Paris, France).

EVs suspensions were diluted in PBS to attain a protein concentration of 0.6 μg per μL and evaluated for size distribution by TEM. Three microliters of the sample were placed on the formvar carbon-coated grid for 5 min and washed with distilled water (three times). For negative contrast the samples were incubated in 2% water solution of uranyl acetate (30 s three times, 5 μl) and left to dry in the small drop (near 1 μl) of last solution. The micrographs were obtained using TEM HITACHI HT 7700 Elexience at 80 kV (with a charge-coupled device camera AMT) and JEM 1011 (JEOL, Japan) equipped with a Gatan digital camera driven by Digital Micrograph software (Gatan, Pleasanton, USA) at 100 kV. The processing of the photos and vesicle size calculation were carried out by ImageJ software. For TEM analysis, 3 different replicates of EVs samples at 4 different stages of the estrous cycle were analyzed.

Aliquots of oEVs samples (one per stage and per side) were fixed with glutaraldehyde 2% for observation under TEM, as previously described [9 (link)]. The micrographs were obtained using TEM HITACHI HT 7700 Elexience at 80 kV (with a charge-coupled device camera AMT) and JEM 1011 (JEOL, Tokyo, Japan) equipped with a Gatan digital camera driven by Digital Micrograph software (Gatan, Pleasanton, CA, USA) at 100 kV. The processing of the photos and vesicle size calculation (min 300 vesicles per stage) were carried out by ImageJ software.