Cultured mammalian cells / Immuno - labeling
1EGFR localization in wet cells
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With use of 40nm gold particles, epidermal growth factor receptors can be seen on fine membranal protrusions. Fully hydrated A431 cells were fixed with 4% paraformaldehyde for 10 minutes, incubated with monoclonal anti-EGFR antibodies and then with 40nm colloidal gold conjugated secondary antibody. The distribution of receptors on the cells is better visualized by counter-staining the cells with 0.1% uranyl acetate.

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2Actin fibers in wet cells labeled with 0.8 nm gold particles
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WETSEMTM Technology is applicable also for intracellular labeling: Fully hydrated CHO cells were fixed with methanol for 5 min at -20C and incubated with mouse anti-actin antibody. Cells were labeled with goat anti-mouse gold particles (0.8 nm ) and the labeling was enhanced with silver staining. Actin fibers are the bright portion in the cytoplasm of the cell.

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3Enzymatic immunostaining for EGFR in wet cells
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Extracellular immunostaining for EGFR using horseradish peroxidase (HRP). Fully hydrated A431 cells were fixed with 4% paraformaldehyde for 15 min. Cells were incubated with monoclonal antibody that recognizes the extracellular epitope of EGFR and then with a secondary antibody conjugated to horseradish peroxidase (HRP). Electron density of precipitate produced by enzymatic reaction of peroxidase with its substrate, DAB, was enhanced by staining with osmium tetroxide. DAB/osmium tetroxide is seen as white precipitate.
Left: Localization of EGFR to membrane is seen in two neighboring cells.
Right: Zoom-in shows localization of EGFR to borders of cellular protrusions.

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4Intracellular labeling of Mitochondria in Hela cells
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Fully hydrated HeLa cells labeled with anti-biotin antibody.
Left: Shows cellular organization of mitochondria in cytoplasm.
Right: In higher magnification (x 12,000) staining of mitochondrial matrix reveals the structure of individual mitochondria.

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