Figure 5. Onset of PC12 process outgrowth.

(A–C) Double immunolabeling of calpain-2 (white or red) and actin (phalloidin, green) in PC12 cells at different stages of differentiation. (A) Calpain-2 labeling in the initial stages of process outgrowth of PC12 cells concentrates around and along the initial buds (arrowheads). (B) Some neurites don't have visible accumulations of calpain-2 (empty arrowheads), and they invariably lack a varicosity (as determined by morphology). (C) Some neurites develop a clear varicone (arrowhead) and calpain-2 labeling concentrates at the varicosity. (D) An example of PC12 cell showing multiple processes with a variety of morphologies. Calpain staining uncovers the varicosity contained in the PC12 terminals, including those in which the presence of a varicosity was not easy to determine by morphology, such as those of triangular shape (asterisk). An incipient process is also highlighted by the presence of calpain-2 puncta (arrowhead in D). Scale bar = 10 µm.