Immuno EM Study of Parafibromin Distribution in Cells

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To complement previous immunolabeling studies in the confocal light microscope, immuno electron microscopy (EM) was performed with the aim of determining the subcellular distribution of the protein parafibromin in human embryonic kidney cells at higher spatial resolution. Parafibromin is implicated in the hyperparathyroidism-jaw tumor familial cancer syndrome, as well as parathyroid cancer and hyperparathyroidism. Cultured cells were introduced into plastic capillary tubes and frozen at 2000 atmospheres in a high-pressure freezing machine to preserve their cellular ultrastructure. The frozen blocks were freeze-substituted in acetone with glutaraldehyde fixative, embedded in resin, sectioned, and labeled with primary antibodies against parafibromin. Specimens were reacted with a secondary antibody conjugated to gold and were imaged digitally in an energy-filtered transmission electron microscope (TEM). Electron micrographs showed that parafibromin was concentrated mainly in the nucleus with some labeling elsewhere in the cell.