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Fig. S2
Upregulatory effect of carrageenan on iNOS protein expression in abdominal tissues of zebrafish.
For immunohistochemistry, after deparaffinization in xylene and rehydration with a graded series of ethanol, endogenous peroxidase activity of the abdominal sections was quenched using 0.3% H2O2 for 30 min. Then, the sections were permeabilized with 0.1% Triton X-100 in PBS for 20 min. Following retrieval of the antigen with proteinase K (20 mM; Sigma) in PBS for 20 min, to decrease nonspecific adsorption we incubated the sections using 5% normal goat serum in PBS for 30 min. The sections were incubated overnight at 4°C with anti-iNOS (1:100 dilution; BD Pharmingen, San Diego, CA, USA; catalog no. 610332) antibody. Finally, after incubation with biotin-conjugated anti-rabbit IgG (1:200 dilution; Vector Laboratories Inc, Burlingame, CA, USA; catalog no. BA-1100) for 30 min followed by avidin-biotin-peroxidase complex for 30 min (Vectastain ABC kit; Vector Laboratories Inc, Burlingame, CA, USA; catalog no. PK-6100), the sections were incubated with 3,32-diaminobenzidine tetrahydrochloride (DAB) (Vectastain ABC kit; Vector Laboratories Inc, Burlingame, CA, USA; catalog no. SK-4100) for 8 min. We analyzed the all stained sections using a Leica DM-6000 CS microscope (Leica Instruments Inc., Wetzlar, Germany) and a microscope digital camera system (SPOT Idea 5 MP CMOS scientific color digital camera system, Diagnostic Instruments, Inc., Sterling Heights, MI, USA). The sections (2 µm) at 24 h after an i.p. injection of vehicle (A) or carrageenan (B). I.p. carrageenan obviously increased iNOS immunoreactivity of the intestine. Scale bars: 100 µm for all images.
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