MVs (15 μg total protein) were first dissolved in PBS and loaded onto UV-treated formvar carbon-coated grids (Ted Pella Inc., Redding, CA, USA). Samples were then fixed in 2% electron microscopy grade paraformaldehyde before being immunostained with anti-CD63 antibody (BD Bioscience, Erembodegem, Belgium) or isotype control (Sigma-Aldrich, St Louis, MO, USA), followed by staining with a 10 nm gold-labelled secondary antibody (Sigma-Aldrich). The samples were then subsequently fixed with 2.5% glutaraldehyde and stained with 2% uranyl acetate. Grids were inserted in a LEO 912AB Omega electron microscope (Carl Zeiss NTS, Jena, Germany) and imaging performed at 70 kV.
Gold labelled secondary antibody
Manufactured by Merck Group
Sourced in United States
The gold-labelled secondary antibody is a laboratory reagent used in immunoassays and other molecular biology techniques. It consists of a secondary antibody conjugated with colloidal gold particles, which serve as a visible label. This product can be used to detect and visualize target proteins or other biomolecules in samples.
Automatically generated - may contain errors
Lab products found in correlation
Isotype control, by Merck Group (1 mentions)
Anti cd63 antibody, by BD (1 mentions)
Leo 912ab omega electron microscope, by Zeiss (1 mentions)
Anti cd63, by BD (1 mentions)
Tecnai f20, by Thermo Fisher Scientific (1 mentions)
Ff300 cu 50, by Electron Microscopy Sciences (1 mentions)
Anti cd9 antibody, by Abcam (1 mentions)
5 protocols using gold labelled secondary antibody
1
Immunostaining and Imaging of Extracellular Vesicles
2
Immunogold Labeling of Exosomes
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Isolated exosomes were loaded onto formwar carbon-coated grids, fixed in 2% paraformaldehyde, washed and immunolabelled with anti-CD63 antibody followed by 10 nm gold-labelled secondary antibody (Sigma Aldrich). The exosomes were post-fixed in 2.5% glutaraldehyde, washed, contrasted in 2% uranyl acetate, embedded in a mixture of uranyl acetate (0.4%) and methyl cellulose (0.13%), and examined by electron microscope (Carl Zeiss NTS).
3
Electron Microscopy of Exosomes
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Exosomes from CSF were loaded onto formwar carbon-coated grids, fixed in 2% paraformaldehyde, washed and immunolabelled with anti-CD63 antibody followed by 10 nm gold-labelled secondary antibody (Sigma Aldrich). The exosomes were post-fixed in 2.5% glutaraldehyde, washed, contrasted in 2% uranyl acetate, embedded in a mixture of uranyl acetate (0.4%) and methyl cellulose (0.13%), and examined by electron microscope (Carl Zeiss NTS).
4
Exosome Immunolabeling for TEM Analysis
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Ten microliters of exosomes from expansion (Passage 6; P6), early (day 3; D3) and late (day 21; D21) osteogenic differentiation of hMSCs respectively were loaded onto formvar carbon-coated grids (Ted Pella Inc, Redding, CA, USA), fixed in 2% formaldehyde, washed and immunolabelled with anti-CD63 (BD Biosciences) antibody followed by 10 nm gold-labelled secondary antibody (Sigma Aldrich, St Louis, MO, USA). The exosomes were post-fixed in 2.5% glutaraldehyde, washed, contrasted with 2% uranyl acetate and air-dried before TEM examination (Tecnai F20, FEI Company, The Netherlands).
5
Visualization of Extracellular Vesicles
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TEM and immuno-TEM analysis of EVs were performed as described previously [4 ] with modifications. Specifically, the vesicle was re-suspended in 1× PBS and deposited onto a formvar-coated slide of copper mesh EM grids (FF300-Cu-50, Electron Microscopy Sciences). The vesicle-coated grids were washed, stained with 1% UO2(CH3COO)2 and then viewed by TEM using a Philips CM12 with Gatan model 791 camera; for the immuno-gold labelling with antibodies, blocked grids coated with EVs were briefly fixed by ice-cold 1:1 ethanol/methanol for 5 min followed by washing with PBS. The grids were then transferred to a drop of the anti-CD9 antibody (Abcam) in PBS with 1% BSA, 0.01% Tween20 and incubated for 30 min. The grids were then washed and incubated with gold-labelled secondary antibody (Sigma). The grids were stained with 0.5% UO2(CH3COO)2 and then imaged by TEM.
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