Cell cultures and tissue biopsies are fixed, cryo-protected and frozen before they are sliced, using a cryo-ultramicrotome (typically a 0.5 mm cell pellet or tissue section is used). The thin slices are incubated with primary and secondary antibodies, lightly stained, and embedded in PVA before tomography is performed.
To analyze 3-D results of in situ samples, the gold-labeled secondary antibody is firstly located on a 2-D electron micrograph. Next, it is subjected to Sidec's unique 3-D reconstruction process to indicate the protein and its neighboring proteins in 3-D. The immunolabeling is combined with inspection of molecular size and characteristic shape features to confirm the identity of a protein. The cell membrane is not visible due to the staining technique used. However, it is reflected as a blank area in the 3-D reconstruction of the cellular material.