Figure 5. Scanning laser confocal immunofluorescent detection of the cytosolic iNOS in primary hepatocyte cultures.

Cell monolayers stimulated with cytokines were permeabilized with digitonin (25 μg/ml) or Triton X-100 (0.1%). A. Image shown in grey scale for the purpose of illustrating cell boundaries and location of the nuclei. Hepatocyte boundaries are indicated with a white dashed line and the nuclei are outlined in red. B. Immunofluorescence was performed with antibodies against the PMP70, EBP50 and iNOS proteins. Green, iNOS; red, EBP50; Blue, PMP70; Magenta, co-localized PMP70 and EBP50; Cyan, co-localized iNOS and PMP70; White, all three proteins colocalized. C. Binding partners in the cytosolic (upper panel, permeabilized with digitonin) and total population (lower panel, permeabilized with Triton X-100) is shown in 2 protein combinations. D. Ultrastructural localization of EBP50 and iNOS in hepatocytes treated with cytokines for 8 hrs. Peroxisomes are identifiable by their urate oxidase crystalline core as well as catalase immunolabeled gold beads (white arrow). As available antibodies shared common source species, EBP50 is shown in two pairings. Catalase (white arrow, 5 nm bead) and EBP50 (black arrow, 10 nm bead). The higher magnification insets panels have immunopositive iNOS (black arrowhead, 5 nm bead) and EBP50 (black arrow, 10 nm bead) localized within a common peroxisome. Experiment is representative of a mixture of cytokine stimulated hepatocytes isolated from 2 separate rats.