Quemesa

Morphological and structural characterization of uncoated and chitosan-coated liposomal vesicles was done using transmission electron microscopy (TEM) employing an EM 208 (Philips) system equipped with a camera (Quemesa, Olympus Soft Imaging Solutions). For this analysis, samples were diluted 1 : 1 with distilled water, then deposited on a Formvar/carbon support film on a specimen grid (Electron Microscopy Sciences). After air-drying for 5 min, the sample was negatively stained with 1% (w/v) of uranyl acetate solution for 10 min.

Morphological characterizations of unloaded and ferrous sulfate–loaded nanoliposomes were performed by transmission electron microscopy (TEM) (EM 208, Philips, Amsterdam, Netherlands) equipped with camera Quemesa (Olympus Soft Imaging Solutions, Münster, Germany). The samples were negatively stained with 1% (w/v) of uranyl acetate solution.
Fe-nanoliposomes were also observed by the optical microscope in fluorescence field Axioplan 2- Image Zeiss, Jena, Germany, equipped with software to capture the images. The Rhodamine B dye was used to visualize lipid vesicles with a 100 X oil immersion objective.

Procedures were performed essentially as described (Raposo et al., 1996 (link); Hurbain et al., 2017 (link)).
For transmission electron microscopy (TEM), sEV preparations were loaded on copper formvar/carbon coated grids (Ted Pella). Fixation was performed with 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), followed by a second fixation with PBS 1% glutaraldehyde in PBS. Samples were stained with 4% uranyl acetate in methylcellulose.
For immunolabeling electron microscopy (IEM), sEV preparations were loaded on grids and fixed with 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Immunodetection was performed with a mouse anti-human CD63 primary antibody (Abcam ab23792). Secondary incubation was next performed with a rabbit anti mouse Fc fragment (Dako Agilent Z0412). Grids were incubated with Protein A-Gold 10 nm (Cell Microscopy Center, Department of Cell Biology, Utrecht University). A second fixation step with 1% glutaraldehyde in PBS was performed. Grids were stained with uranyl acetate in methylcellulose.
All samples were examined with a Tecnai Spirit electron microscope (FEI, Eindhoven, The Netherlands), and digital acquisitions were made with a numeric 4k CCD camera (Quemesa, Olympus, Münster, Germany). Images were analyzed with iTEM software (EMSIS) and statistical studies were done with Prism-GraphPad Prism software (v8).

Cells were fixed with a mixture of 2% (w/v) paraformaldehyde and 0.2% (w/v) glutaraldehyde in 0.1 M PHEM buffer (120 mM PIPES, 50 mM HEPES, 4 mM MgCl₂, 20 mM EGTA pH 6.9), and processed for immunoelectron microscopy as described previously (Setty et al., 2007 (link)). Ultrathin cryosections were prepared using UC7 ultracryomicrotome (Leica, Vienna, Austria) (Raposo et al., 1997 ), double immunogold labeled with protein A conjugated to 10- or 15-nm gold particles (PAG10, PAG15 from Cell Microscopy Center, AZU, Utrecht, The Netherlands) and analyzed under a Tecnai Spirit electron microscope (FEI, Eindoven, The Netherlands) equipped with a 4k CCD camera (Quemesa, Olympus).

Procedures were performed essentially as described [25 (link),32 (link),33 (link)]. Immunodetection was carried out with the following primary antibodies: mouse anti-human CD63 (Abcam ab23792), mouse anti-human CD9, or mouse anti-human CD81 (both from Dr E. Rubinstein, Université Paris-Sud, Institut André Lwoff, Villejuif, France). Secondary incubation was performed with a rabbit anti mouse Fc fragment (Dako Agilent Z0412), then grids were incubated with Protein A-Gold 10 nm (Cell Microscopy Center, Department of Cell Biology, Utrecht University). All samples were observed with a Tecnai Spirit electron microscope (FEI, Eindhoven, The Netherlands), and digital acquisitions were made with a numeric 4k CCD camera (Quemesa, Olympus, Münster, Germany). Images were analysed with iTEM software (EMSIS) and statistical studies were done with GraphPad Prism software (v8).

Pf4* phages were resuspended in PBS and a drop of suspension was placed on a petri dish. A Formvar EM grid was placed Formvar side down on top of the phages drop for approximately 1 min. The grid was removed, blotted with filter paper, and placed onto a drop of 7.0% uranyl acetate in distilled water for 3 min. The grid was washed in water, blotted again with filter paper, and examined on EM JEOL 1010 at 80 KV. The pictures were recorded by using Quemesa, the electron digital camera of Olympus.