Localization of gelatinolytic activity in fibrotic liver tissue. (A and B) Collagenous septa in fibrotic rat liver tissue were stained with Sirius red with rectangular detail that highlights collagen fibers in (B). Original magnification, ×20. (C and D) In situ zymography localizes strong gelatinolytic activity to fibrous structures with rectangular detail that demonstrates nonpericellular, fiber-associated gelatinase activity in (D). Original magnification, ×40. (E-H) Cryostat sections of fibrotic human liver tissue were incubated with 25 ng of proMMP-2 (F) or proMMP-9 (H) or were left untreated (E and G). Bound matrix metalloproteinases (MMPs) were detected using immunohistochemistry monoclonal antibodies specific for MMP-2 (arrows in E and F depict endogenous MMP expression) or MMP-9 (G and H). An irrelevant primary antibody served as a control (I). Sections shown represent three independent experiments. Scale bars, 100 μm.