**2.4.2 Preparation of animal tissues for IHC**


8-Nitroguanine, a Potential Biomarker to

min at room temperature.

5% (w/v) skim milk.

Evaluate the Risk of Inflammation-Related Carcinogenesis 207

4. The sections are incubated with 3% H2O2 for 30 min, and rinse sections in PBS for 10

5. Block sections by 30 min incubation in PBS containing 5% (v/v) normal goat serum or

6. The sections are incubated with the primary antibody, rabbit polyclonal anti-8-

8. The sections are incubated with goat anti-rabbit IgG antibody (1:200) for 3h at room

9. The sections are incubated with peroxidase anti-peroxidase complex (PAP, 1:200) for 2h

11. Incubate sections in DAB/Tris solution 10 mg 3,3-diaminobenzidine tetrahydrochloride dihydrate in 100 ml 0.05 M Tris, pH 7.4; filter under a hood with general usage filter paper. Incubate sections in developer for up to 15 min. Check sections frequently under

16. Dehydrate sections in Coplin jars containing graded ethanol 50–80%, 3 min each; twice in 95% ethanol 3 min each; and 3 times in 100% ethanol 3 min each. Tissue is cleared by incubation 3 times for 5 min each in xylene, and coverslipped using Maninol mounting

To prepare pre-absorbed 8-nitroguanine antibody place equal amounts of 8-nitroguanine antibody in 1.5 ml Eppendorf microfuge tubes. For the primary antibody to be pre-absorbed pure 8-nitroguanine is added to give a final protein concentration greater than 1 g/ml. An equal amount of diluents without 8-nitroguanine is added to the control antiserum. Mixtures are incubated for 2 h at room temperature and then diluted to their final working concentration with antibody buffer. Pre-absorption is then continued by incubation overnight at 4°C prior to use. Control and pre-absorbed antibody are used in parallel at

a microscope for the degree of development, and reaction product is brown. 12. Terminate reaction by washing sections 3 times for 10 min each in 0.05 M Tris, pH 7.4.

nitroguanine antibody (1–2 g/ml), overnight at room temperature. 7. Wash sections in PBS at room temperature, 3 times for 5 min each.

at room temperature, fellow by wash in PBS, 3 times for 5 min each. 10. Equilibrate sections in 0.05 M Tris, pH 7.5 with two washes for 10 min each.

14. Counterstain sections with Mayer hematoxylin for 1-1.5 min if necessary.

**3. 8-Nitroguanine accumulation in inflammation-related cancer** 

**3.1 Application of Immunochemistry employing anti-8-nitroguanine antibody** 

We have performed immunohistochemical analysis for 8-nitroguanine formation in various clinical specimens and animal models of inflammation-related carcinogenesis. We have firstly demonstrated that 8-nitroguanine is formed at the sites of carcinogenesis regardless of etiology, and we have proposed the possibility that 8-nitroguanine is a potential biomarker to evaluate the risk of inflammation-associated carcinogenesis (Kawanishi & Hiraku, 2006; Kawanishi et al., 2006). In clinical specimens, 8-nitroguanine was formed in the gastric grand epithelial cells of patients with gastritis caused by *Helicobacter pylori* (*H. pylori*)

temperature, fellow by wash in PBS, 3 times for 5 min each.

13. Wash sections 2 times for 5 min in distilled water (DW).

15. Wash sections 2 times for 5 min in DW.

Step C. Pre-absorption immunostaining

StepA or StepB. Proceed as StepA (1) through (10).

medium.

used. For a mouse approximately 80 ml over 5 min of fixative should be infused. After completion of the procedure, the arms and tip of the nose should be stiff.


### **2.4.3 Detailed procedure for IHC staining**

Step A. Immunofluorescent staining procedure


#### Step B. Peroxidase anti-peroxidase immunohistochemical method


10. Disconnect the rat or mouse from the perfusion apparatus and carefully remove the organ to avoid damage to the tissue. After carefully removing organ from the body,

11. Following incubation, tissue for IHC should be paraffin-embedded and then sectioned at 5m thickness onto silanized glass slides. Slides may be stored at ambient

1. Paraffin sections of human or animal tissues are deparaffinized in xylene for 3 min with frequent shaking in a glass box. Then, the sections are treated in xylene in another glass box for 3 min, followed by the treatment with 100, 90, 80, 70, and 50% (v/v) ethanol for 30–60 sec. To insure complete removal of paraffin, soak sections in PBS for 30 min. 2. To retrieve the antigens, the sections are heated in 5% (w/v) urea for 5 min in a microwave oven, and then left sections on the 5% urea until the temperature reducing

4. Block sections by 30 min incubation in PBS containing 5% (v/v) normal goat serum

5. The sections are incubated with the primary antibody, rabbit polyclonal anti-8 nitroguanine antibody (1–2g/ml), overnight at room temperature. When double immunofluorescence labeling study is performed, mouse monoclonal antibody is mixed with anti-8-nitroguanine antibody and the sections are treated with this mixture. Note: the final concentration of antibody depends on the specific antibody preparation being

7. The sections are incubated with the secondary antibody, Alexa 594-labeled goat antibody against rabbit IgG (1:400) for 3 h at room temperature. When double immunofluorescence labeling study is performed, Alexa 488-labeled goat antibody against mouse IgG (1:400) is mixed with Alexa 594-labeled goat antibody against rabbit IgG and the sections are treated with this mixture. Note: if primary serum is derived from a source other than rabbit, then the choice of secondary antibody should be

9. Pipet Dapi-Fluoromount-GTM Mounting Medium onto the section and then cover with a

6. The sections are washed with PBS at room temperature, 3 times for 5 min each.

completion of the procedure, the arms and tip of the nose should be stiff.

continue fixation of adult tissue at room temperature for 3 h.

3. Rinse sections in PBS at room temperature, 3 times for 5 min each.

temperature in slide cases until use.

**2.4.3 Detailed procedure for IHC staining** 

to room temperature.

Step A. Immunofluorescent staining procedure

antibody buffer, or 5% (w/v) skim milk.

adjusted accordingly for specificity. 8. Wash sections with PBS 3 times over 30 min.

1. Deparaffinize as Step A (1)

2. To retrieve the antigens as Step A (2).

used and may need to be empirically determined.

cover glass. Dry overnight covered at 4°C on a refrigerator. 10. The stained sections are examined under a fluorescent microscope.

Step B. Peroxidase anti-peroxidase immunohistochemical method

3. Rinse sections in PBS at room temperature, 3 times for 5 min each.

used. For a mouse approximately 80 ml over 5 min of fixative should be infused. After


#### Step C. Pre-absorption immunostaining

To prepare pre-absorbed 8-nitroguanine antibody place equal amounts of 8-nitroguanine antibody in 1.5 ml Eppendorf microfuge tubes. For the primary antibody to be pre-absorbed pure 8-nitroguanine is added to give a final protein concentration greater than 1 g/ml. An equal amount of diluents without 8-nitroguanine is added to the control antiserum. Mixtures are incubated for 2 h at room temperature and then diluted to their final working concentration with antibody buffer. Pre-absorption is then continued by incubation overnight at 4°C prior to use. Control and pre-absorbed antibody are used in parallel at StepA or StepB. Proceed as StepA (1) through (10).
