Fig. 4. Tight junction protein expression in the paw tissue. (A ) Paw tissue from untreated rats was obtained and stained for claudin-1 (red ) or claudin-5 (red ) and occludin (green ) as well as diamidinophenylindole (DAPI) for nuclei (blue ) (magnification 10×). Different tissue types, such as epidermis (B ), and peripheral nerves and blood vessels (C ) known to express claudin-1 were identified by morphology based on DAPI nuclear staining within the structure (magnification 40×). (D ) Claudin-5 immunoreactivity was observed in blood vessels, but not in the perineurium of the peripheral nerve (arrows ) (magnification 40× and 100×, bar = 50 and 10 μm, respectively). (E, F ) Paw tissue from normal rats was double-stained for claudin-1 (green ) and calcitonin gene-related peptide (red ) as well as DAPI for nuclei (blue ) to identify sensory nerves in comparison with blood vessels. One representative example of blood vessels (E ) and of peripheral nerves (F ) is shown (magnification 100×, bar = 10 μm). (F ) Negative control by omission of the primary antibody (anti claudin-1 antibody) is shown on the right (magnification 100×, bar = 10 μm). CGRP = calcitonin gene-related peptide; DAPI = diamidinophenylindole.