Fei xl30 scanning electron microscope

The specimens of cellulite (10 living female) were fixed with glutaraldehyde 2 % in 0.1 M PB, post-fixed in 1 % osmium tetraoxide (OsO₄) in the same buffer for 1 h, dehydrated in concentrations of acetone, critical point dried (CPD 030, Balzers, Vaduz, Liechtenstein), fixed to stubs with colloidal silver, sputtered with gold by an MED 010 coater (Balzers, Brugherio, Italy), and examined with an FEI XL30 scanning electron microscope (FEI Company, Eindhoven, Netherlands).
The analysis of SEM/TEM images has been done by two independent experts morphologist, blinded for the gender of the samples.

Upon washing with 0.1 M phosphate buffer, the lens capsule was dehydrated in a graded series of ethanol with 20% increase in concentration every 15 min followed by hexamethyldisilazane (HMDS) treatment, 25% increments every 30 min. Sample was allowed to air dry overnight, and subsequently sputter-coated with gold–palladium film. SEM images were obtained using a Philips FEI-XL30 scanning electron microscope (FEI Company, CA, USA).

As described previously [12 (link),13 (link)], exosome samples were fixed overnight in 4% paraformaldehyde in 0.1M cacodylate buffer at PH 7.4. On a carbon–Formvar-coated 200 mesh nickel grid, five microliters (5 µL) of suspended exosome preparation was applied and allowed to stand 30 min. Whatman filter paper was used to wick off the excess sample. With the exosome side down, grids were floated on a 20 µL drop of 1M ammonium chloride for 30 min to quench aldehyde groups from fixation steps. Afterward, grids were floated on drops of blocking buffer (0.4% BSA in PBS) for 2 h and then rinsed 3 times for 5 min each with PBS. Grids were set up as follows and were allowed to incubate in blocking buffer or primary antibody (anti-TGFb, anti-IL10, or anti-CD63) for 1 h. For 1 h, grids were floated on drops of 1.4 nm nanogold secondary antibody (Nanoprobes, Inc., Yaphank. NY, USA), which was diluted at 1:1000 in blocking buffer. Grids were then rinsed 3 times for 5 min each with DI H2O. Exosome samples were postfixed in 2% osmium tetroxide in NaCac buffer and dehydrated in ethanol for visibility in electron SEM. Then, samples were mounted on aluminum stubs and sputter-coated for 6 min with gold–palladium (Anatech Hummer 6.2, Union City, CA, USA). Using an FEI XL30 scanning electron microscope (FEI, Hillboro, OR, USA), exosomes were observed and imaged at 10 kV.