Negative Staining of Extracellular Vesicles

Samples were prepared using a two-step protocol for negative staining^{64 (link)}. The sEV suspensions were adsorbed for 30 min onto carbon-coated 300 mesh copper grids (Electron Microscope Science, Hatfield, PA, USA), washed three times with pure water, and then stained for 30 s with an uranium free staining solution^{65 (link)}. The grids were then paper-drained and directly analysed with a Libra 120 Plus transmission electron microscope, operating at 120 kV and equipped with an in-column omega filter and 16-bit CCD camera (Zeiss, Oberkichen, Germany). Samples were analysed with ImageJ software (NIH).

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Anastasi F., Greco F., Dilillo M., Vannini E., Cappello V., Baroncelli L., Costa M., Gemmi M., Caleo M, & McDonnell L.A. (2020). Proteomics analysis of serum small extracellular vesicles for the longitudinal study of a glioblastoma multiforme mouse model. Scientific Reports, 10, 20498.

Publication 2020

Libra 120 Plus 16-bit CCD camera

Carbon Copper Electron microscope Transmission electron microscope Uranium

Corresponding Organization :

Other organizations : International Flame Research Foundation, Scuola Normale Superiore, Scuola Superiore Sant'Anna, Fondazione Umberto Veronesi, Center for Nanotechnology Innovation, Italian Institute of Technology, Fondazione Stella Maris, University of Padua

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Variable analysis

independent variables

Preparation of samples using a two-step protocol for negative staining

dependent variables

Morphological characteristics of small extracellular vesicles (sEVs) observed under transmission electron microscope

control variables

Carbon-coated 300 mesh copper grids used for adsorption of sEV suspensions
Washing of grids three times with pure water
Staining of grids for 30 s with an uranium free staining solution
Transmission electron microscope (Libra 120 Plus) operating at 120 kV and equipped with an in-column omega filter and 16-bit CCD camera
Analysis of samples using ImageJ software

controls

No positive or negative controls were explicitly mentioned in the provided information.

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