**2.2 Specificity of anti-8-nitroguanine antibody**

Specificity of the purified antibody was examined by a dot immunobinding assay and absorption test (Pinlaor et al., 2004a). Purified antibody gave a strong immunostaining only on the spot of 8-nitroguanine conjugate (Fig. 2A). The immunoreactivity disappeared only when the antibody was pre-incubated with 8-nitroguanine. In contrast, immunoreactivity with 8-nitroguanine conjugate did not disappear when the antibody was preincubated with 3-nitrotyrosine, guanosine, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), deoxyguanosine, 8-bromoguanosine, and xanthosine (Fig. 2B).

Fig. 2. Dot immunobinding assay and absorption test of anti-8-nitroguanine antibody.

8-Nitroguanine, a Potential Biomarker to

Paraformaldehyde (Nacalai, Kyoto, Japan)

Sliding microtome (RM2265, Leica, Germany)

**2.4.2 Preparation of animal tissues for IHC** 

6. Make a small incision in the right atrium.

is no response, then proceed with the surgery.

side of the animal to allow proper drainage of perfusate.

Ethanol (Nacalai, Kyoto, Japan) Xylene (Nacalai, Kyoto, Japan)

**2.4 Detailed procedure for IHC** 

pentobarbital.

forceps.

respectively.

end of this step.

give access to the heart.

Evaluate the Risk of Inflammation-Related Carcinogenesis 205

Monobasic and dibasic salts of sodium and potassium phosphate (Nacalai, Kyoto, Japan)

Average time for immunohistochemical (IHC) localization of 8-nitroguanine is 2-3 days, not including preparation and mounting of tissues. Albeit, time varies with duration of antibody incubation of second antibody in double fluorescent immunohistochemistry.

1. Animal is weighed and anesthetized by intraperitoneal injection of 50 mg/kg sodium

2. After the animal has fallen asleep, firmly pinch the foot with a pair of tweezers. If there

3. Position the animal on its back. Open the abdominal cavity with a midline incision to the sternum. Make a diagonal cut to each side of the sternum through the rib cage extending to either side of the neck. Care should be taken not to sever any vessels or puncture the heart. The sternum may now be clamped back with hemostatic forceps to

4. Cut the connective tissue surrounding the diaphragm and make a lateral cut on each

5. Lift the lungs to expose the descending aorta and occlude the vessel using hemostatic

7. Insert an i.v. catheter into the left ventricle. Remove the needle from the catheter. Position so that the tip of the catheter resides within the aortic arch. The optimal size of the catheter for transcardial perfusion is 18 and 22 gauge for rat and mouse,

8. Connect the catheter to the perfusion apparatus and begin the flow of 0.9% NaCl into the animal. For an adult rat 80 ml 0.9% saline should be infused over 3 min. For a mouse 20 ml saline should be infused over 2 min. Progress of the perfusion may be monitored by assessment of the eyes and gums which should be blanched toward the

9. Switch perfusion to 0.1 M phosphate buffer (PB) containing 4% paraformaldehyde. It is common to see turgor and twitching of the upper extremities at the initial flow of fixative into the animal. Perfuse an adult rat, 300 ml of fixative over 10 min should be

Confocal laser scanning microscopy (FV-1000D, Olympus, Tokyo, Japan)

**2.4.1 Time required for immunohistochemistry of 8-nitroguanine** 

Fluorescent light microscope (BX53, Olympus, Tokyo, Japan)

Antigen–antibody reactions were visualized by the peroxidase-anti-peroxidase method. Purified antibody gave a strong immunostaining only on the spot of 8-nitroguanine conjugate (A). 8-Nitroguanine, 3-nitrotyrosine, guanosine, 8-oxodG and deoxyguanosine were incubated with the antibody at the concentration of 0.7 g/ml, and were applied to 8 nitroguanine conjugate. Immunoreactivity disappeared only when the antibody was pretreated with 8-nitroguanine (B). 8-NG, 8-nitroguanine; 3-NT, 3-nitrotyrosine; G, guanosine; dG, deoxyguanosine and 8-BromoG, 8-bromoguanosine.
