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. Author manuscript; available in PMC: 2014 Jun 24.
Published in final edited form as: Methods Cell Biol. 2010;96:619–648. doi: 10.1016/S0091-679X(10)96026-3

Figure 3. Immuno-labeling of VSVG using HPF/FS and Lowicryl embedding.

Figure 3

A: Flow diagram of a typical HPF/FS experiment using Lowicryl HM20 embedding and immunolabeling (see section III.2.c. for details). B: Electron micrograph of immunogold labeling for GFP in a HeLa cell after HPF/FS and Lowicryl HM20 embedding. HeLa cells grown on sapphire discs were transfected with a construct expressing a temperature-sensitive VSVG-GFP. With the temperature block present the protein accumulates in the Golgi complex (G). 30 min after release of the temperature block the cells were high-pressure frozen and processed. Most of the protein has reached the cell surface but there is still protein present inside the Golgi and in vesicular profiles near the Golgi (arrows). This procedure preserves antigenicity as well as structural elements such as microtubules (arrowhead), which are very sensitive to chemical fixation. Scale bar represents 500 nm.