Negative Staining for Small Extracellular Vesicles

To visualize small EVs, the fractions were negatively stained. For negative staining, a glow-discharged carbon-coated (2 nm carbon) formvar grid was placed on a 20 μL drop of sample and allowed to adsorb to the carbon for 10 s. The sample was then washed three times briefly on a drop of water, stained with 3% w/v uranyl acetate in water, and dried. Micrographs were recorded using a transmission electron microscope (JEM 1400; JEOL Ltd, Freising, Germany) with a bottom-mounted high-sensitivity 4K CMOS camera (TemCam F416; TVIPS, Gauting, Germany) [60 (link)].

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Lajqi T., Köstlin-Gille N., Hillmer S., Braun M., Kranig S.A., Dietz S., Krause C., Rühle J., Frommhold D., Pöschl J., Gille C, & Hudalla H. (2022). Gut Microbiota-Derived Small Extracellular Vesicles Endorse Memory-like Inflammatory Responses in Murine Neutrophils. Biomedicines, 10(2), 442.

Publication 2022

JEM 1400 TemCam F416

Carbon Cmos Formvar Sensitivity Transmission electron microscope Uranyl acetate

Corresponding Organization : University Hospital Heidelberg

Other organizations : University of Tübingen, Zentrum für Kinderheilkunde

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Variable analysis

independent variables

Negative staining of small EVs

dependent variables

Visualization of small EVs

control variables

Glow-discharged carbon-coated (2 nm carbon) formvar grid
20 μL drop of sample
10 s adsorption time
3 brief washes on a drop of water
Staining with 3% w/v uranyl acetate in water
Transmission electron microscope (JEM 1400; JEOL Ltd, Freising, Germany)
Bottom-mounted high-sensitivity 4K CMOS camera (TemCam F416; TVIPS, Gauting, Germany)

controls

No positive or negative controls were explicitly mentioned in the input protocol.

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