Figure 4. Tbdn and ZO-1 proteins show reciprocal regulation in cold-adapted smelt rete and choroidal blood vessels. Compared to warm fish maintained at 8–10 °C (A: warm specimen/Tbdn stain; C: warm specimen/ZO-1 stain), the endothelial linings of the choroidal (c) and rete (r) blood vessels of cold fish maintained at 0.5 °C show a higher expression level of Tbdn protein, but a lower level of ZO-1 protein in these regions (B: cold specimen/Tbdn stain; D: cold specimen/ZO-1 stain). The arrows indicate choroidal and rete blood vessel endothelia. E: Sections were also incubated with a control IgG and showed no staining of the blood vessels. Positive staining for Tbdn (OE5) and ZO-1 appears as bright red staining. The dark brown or black color in all panels is intrinsic due to pigmentation from the pigments cells of the choroidal vasculature and/or retinal tissue. The immunohistochemical results shown here are representative of three smelt in each of the warm and cold groups and are quantitated in Figure 6. The scale bar in the lower right corner of the figure indicates 100 μm.