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First published online May 2, 2008
Journal of Experimental Biology 211, 1524-1534 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.014894
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Occludin immunolocalization and protein expression in goldfish

Helen Chasiotis* and Scott P. Kelly

Department of Biology, York University, Toronto, ON, Canada, M3J 1P3


Figure 1
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  		Fig. 1. Immunofluorescence staining of (A) Na+,K+-ATPase (green) and (B) occludin (red) in longitudinal sections of a goldfish gill filament. Nuclei are stained with DAPI (blue) in A and F. C is a merged image of A and B. In A, Na+,K+-ATPase immunolocalizes to select cells within the interlamellar (IL) region of the primary filament and at the base of secondary gill lamellae. In B, discontinuous occludin immunostaining is concentrated along the edges of secondary gill lamellae (white arrow), including medial parts of lamellae that are embedded within the body of the primary filament, and is associated with cells that also stain for Na+,K+-ATPase (white arrowhead). Occludin immunostaining is also prominent in parts of the IL region that line the lateral walls of the central venous sinus (CVS). D and E show, at higher magnification, areas from C that are indicated by the white arrowhead and white arrow, respectively. Control sections probed with secondary antibody only show DAPI fluorescence (F). Scale bars in A, B, C and F are 20 µm. Scale bars in D and E are 5 µm.

 

Figure 2
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  		Fig. 2. Immunofluorescence staining of (A,B) Na+,K+-ATPase(green) and (A,C) occludin (red) in cross-sections of goldfish intestine. D is a merged image of B and C. In A, immunostaining for Na+,K+-ATPase is concentrated to basolateral regions and occludin immunostaining is apparent in apical regions of epithelia lining the intestinal lumen (L). Occludin immunostaining appears less prominent in epithelial cells at the base of the intestinal villi (see white arrows in A). At higher magnification (see dashed box in A, which outlines area magnified in C,D), occludin immunostaining appears as a typical honeycomb-like tight junction (TJ) pattern. Nuclei are stained with DAPI (blue), and only DAPI staining is observed in sections probed with secondary antibody alone (E). All scale bars are 20 µm.

 

Figure 3
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  		Fig. 3. (A) Schematic illustration of a goldfish nephron based on morphological criteria previously outlined (Sakai, 1985Go). The goldfish nephron can be divided into the following regions: 1, renal corpuscle (Bowman's capsule and glomerulus); 2, proximal tubule; 3, distal tubule; and 4, collecting duct. (B) Cross-section of goldfish kidney immunostained for occludin (red) and Na+,K+-ATPase (green). Occludin and Na+,K+-ATPase show region-specific immunostaining along the nephron. Numbered labels indicate regions of the nephron as defined in A. No occludin or Na+,K+-ATPase immunostaining was found in the renal corpuscle. A control section can be seen in C (i.e. DAPI fluorescence only). All scale bars are 20 µm.

 

Figure 4
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  		Fig. 4. Immunofluorescence staining of (A–C) Na+,K+-ATPase (green) and (D–F) occludin (red) in cross-sections of goldfish proximal tubule (A,D,G), distal tubule (B,E,H) and collecting duct (C,F,I). Nuclei are stained with DAPI (blue) in A–C. G, H and I are merged images of A and D, B and E, and C and F, respectively. Immunostaining for Na+,K+-ATPase is restricted primarily to the basal membrane of renal epithelial cells in the proximal region of the nephron (A) and to the basolateral membrane of renal epithelial cells in distal and collecting segments (B,C). No occludin immunostaining is observed in proximal segments of the nephron (D). Strong and moderate occludin immunostaining is concentrated at the apical membrane of renal epithelial cells lining the lumen of the distal tubule and collecting duct, respectively (E,F). All scale bars are 20 µm.

 

Figure 5
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  		Fig. 5. Western blot analysis of occludin expression in goldfish gill, intestine and kidney. Single occludin immunoreactive bands (at ~68 kDa) always appeared for goldfish epithelial tissue. A non-epithelial negative control tissue (goldfish blood cells) was not immunoreactive. A positive tissue control (rat kidney) was found to resolve at ~65 kDa.

 

Figure 6
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  		Fig. 6. Effects of 1, 2 and 4 weeks feeding and food deprivation on relative mass gain/loss in goldfish. Data are expressed as mean values ± s.e.m. (N=10 per group). *Significant difference (P<0.05) between fed and unfed groups at the same time point. {dagger}Significant difference (P<0.05) from 1 week unfed group. {ddagger}Significant difference (P<0.05) from 2 weeks unfed group.

 

Figure 7
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  		Fig. 7. Effects of 1, 2 and 4 weeks feeding and food deprivation on (A) serum osmolality, (B) serum Na+, (C) serum Cl– and (D) muscle water content in goldfish. Data are expressed as mean values ± s.e.m. (N=10 per group). *Significant difference (P<0.05) between fed and unfed groups at the same time point. {dagger}Significant difference (P<0.05) from 1 week unfed group.

 

Figure 8
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  		Fig. 8. Effects of 1, 2 and 4 weeks feeding and food deprivation on (A) gill Na+,K+-ATPase activity and (B) normalized gill occludin expression in goldfish determined by Western blot analyses. (C) Representative Western blot of gill occludin protein expression in fed and unfed goldfish. Data are expressed as mean values ± s.e.m. (N=8–10 per group). *Significant difference (P<0.05) between fed and unfed groups at the same time point. {dagger}Significant difference (P<0.05) from 1 week unfed group.

 

Figure 9
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  		Fig. 9. Effects of 1, 2 and 4 weeks feeding and food deprivation on (A) intestine Na+,K+-ATPase activity and (B) normalized intestine occludin expression in goldfish determined by Western blot analyses. (C) Representative Western blot of intestine occludin protein expression in fed and unfed goldfish. Data are expressed as mean values ± s.e.m. (N=8–10 per group). *Significant difference (P<0.05) between fed and unfed groups at the same time point. {dagger}Significant difference (P<0.05) from 1 week unfed group. {ddagger}Significant difference (P<0.05) from 2 weeks unfed group.

 

Figure 10
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  		Fig. 10. Effects of 1, 2 and 4 weeks feeding and food deprivation on (A) kidney Na+,K+-ATPase activity and (B) normalized kidney occludin expression in goldfish determined by Western blot analyses. (C) Representative Western blot of kidney occludin protein expression in fed and unfed goldfish. Data are expressed as mean values ± s.e.m. (N=8–10 per group). *Significant difference (P<0.05) between fed and unfed groups at the same time point. {dagger}Significant difference (P<0.05) from 1 week unfed group. {ddagger}Significant difference (P<0.05) from 2 weeks unfed group.

 

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© The Company of Biologists Ltd 2008