WETSEM® Gallery
Cultured mammalian cells Immunolabeling
Receptor
patterning in wet cells
Epidermal growth factor receptor labeling. Fully hydrated A431 cells were fixed with 2% paraformaldehyde for 7 min and incubated with mouse anti-EGFR. Labeling was done using 20 nm colloidal gold conjugated to anti-mouse IgG secondary antibody and silver enhancement.
Upper: At low magnification membranal staining can be seen on cell border.
Lower: Higher magnification of the marked area in the left image shows patterning of individual gold particles.
(In collaboration with Prof. Joseph Schlessinger, Yale University)
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EGFR
localization in wet cells
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 relative to the cells is shown by conterstaining the cells with 0.1% uranyl acetate.
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Actin
fibers in wet cells labeled with 0.8 nm gold particles
QX Technology is applicable also for intracellular labeling: Fully hydrated CHO cells were fixed with methanol for 5 min at -20°C 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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Enzymatic
immunostaining for EGFR in wet cells
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 recognize 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.
Upper: Localization of EGFR to membrane is seen in two neighboring cells.
Lower: Zoom-in shows localization of EGFR to borders of cellular protrusions.
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Mitochondria
stained with anti-biotin antibodyin wet cells
Fully hydrated HeLa cells stained with anti-biotin antibodies, which label predominately mitochondrial enzymes. The cells were fixed with 4% paraformaldehyde for 15 minutes and permeabilized with 0.2% Triton X-100. Cells were incubated with monoclonal anti-biotin antibody and labeled with secondary antibody conjugated to 0.8nm gold particles. Gold labels were enhanced using silver.
Upper: Shows Cellular organization of mitochondria in cytoplasm.
Lower: In higher magnification (x 12,000) staining of mitochondrial matrix reveals the structure of individual mitochondria.
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Product Information
For more information on WETSEM® capsules for imaging and EDS of fully-hydrated samples with SEM, please see the Product information.
