HeLa cell endocytic pathway labeled for EGFR and transferrin

Autor:

Matthew R G Russell

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https://czwiki.cz/b/9279

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Compartments of the endocytic pathway in HeLa cells. Early endosomes (E), late endosomes/MVBs (M), and lysosomes (L) are visible. Epidermal growth factor receptors (EGFR) and transferrin (Tf) are labelled in the early endosomes.

Epidermal growth factor receptors are labelled with an antibody conjugated to 10 nm gold, applied to the cell surface. The cells were stimulated with EGF and allowed to internalize the receptors, bringing the gold with them. The image was taken 5 minutes after internalization, at which point most of the EGFR-gold has reached early endosomes (black dots, marked by arrowheads), but has not yet entered late endosomes or lysosomes. The cells are also loaded with transferrin conjugated to horseradish peroxidase (TfHRP). The HRP catalyses a reaction in the presence of DAB that produces a dark stain in the transferrin containing compartments in the image.

HeLa cells were preincubated for 1 h in serum-free medium. For the final 30 minutes the cells were incubated with TfHRP. The cells were then incubated with EGF and anti-EGFR 10 nm gold-conjugated antibody for 30 minutes at 4°C, washed, and incubated at 37°C for a further 5 minutes, all in the presence of TfHRP. Arrowheads; anti-EGFR 10 nm gold. Dark content; cross linked TfHRP. Bar, 500 nm.

Philips EM400 TEM

Methods for cell fixation and preparation for electron microscopy can be found in the reference below. Briefly, cells were room temperature fixed, a DAB reaction was done, and the cells were osmium stained, dehydrated and embedded en face in epon.

Related reference;

Doyotte, A., Russell, M.R.G., Hopkins, C.R., Woodman, P.G. (2005) Depletion of TSG101 forms a mammalian ‘‘Class E’’ compartment: a multicisternal early endosome with multiple sorting defects. J. Cell Sci, 118:3003-3017.

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