Granules containing endogenous NPY or tPA do contain CgA. Cultured bovine chromaffin cells were fixed with 4% paraformaldehyde and permeabilized with methanol. (A) Cells were incubated with antibodies to NPY and bovine CgA, followed by Alexa Fluor–labeled anti-mouse and anti-rabbit secondary antibodies, and then visualized by confocal microscopy. (B) Cells were incubated with a primary antibody to tPA, followed by Alexa₄₈₈-labeled goat anti-rabbit Fab fragments, and then blocked with an excess of unlabeled goat anti-rabbit Fab fragments. Cells were then incubated with or without rabbit anti-bovine CgA, followed by an Alexa₅₄₆-labeled anti-rabbit secondary antibody, and imaged by confocal microscopy. The absence of significant immunofluorescence in cells with no second primary antibody against CgA (blue line in B; n = 8 cells, 481 puncta) indicates that the first rabbit primary antibody against tPA was completely blocked before the addition of the second primary. Endogenous CgA in tPA-containing granules, n = 11 cells, 883 puncta. Endogenous CgA in granules in cells without tPA, n = 12 cells, 392 puncta.