Figure 1.

PAI colocalizes with endogenous tPA in secretory granules. (A, C, and E) Cultured bovine chromaffin cells were fixed with 4% paraformaldehyde, permeabilized with methanol, and incubated with a primary antibody to tPA (rabbit anti-mouse tPA; Molecular Innovations), followed by Alexa Fluor 488–labeled goat anti–rabbit Fab fragments (Jackson ImmunoResearch Laboratories). Fab fragments rather than bivalent antibodies were used to preclude capture of a second rabbit primary antibody in a subsequent labeling step. After rinsing, the cells were blocked with an excess of unlabeled goat anti–rabbit Fab fragments (Jackson ImmunoResearch Laboratories), to ensure that none of the first primary ab (rabbit anti-tPA) would be accessible to a second anti-rabbit secondary antibody. (B, D, and F) Cells were next incubated with (B, D) or without (F) rabbit anti–human PAI (Abcam), followed by an Alexa Fluor 546–labeled anti-rabbit secondary antibody (B, D, and F; Molecular Probes). Cells were imaged by confocal microscopy. Images to be compared directly (e.g., A and E; B and F) were acquired at the same microscope settings, and the brightness and contrast were adjusted identically in making the figures. The absence of immunofluorescence in F (with no second primary antibody against PAI) indicates that the first rabbit primary antibody visualized in E was completely blocked before the addition of the second primary (seen in B and D). Colocalization of PAI (B) and tPA (A) is indicated by arrowheads and at an expanded scale in D and C, respectively. Bars, 2 µm.