**2.3.2 Chemicals and reagents for immunohistochemistry (IHC) and special equipment**

Silanized slides (Dako, Japan)

Dapi-Fluoromount-GTM Mounting Medium (Southern Biotech, Birmingham, USA)

Malinol mounting medium (Muto Pure Chemicals Co. Tokyo,Japan)

3,3'-Diaminobenzidine tetrahydrochloride dehydrate (DAB) (Dojindo, Kumamoto, Japan)

Goat anti-rabbit -globulin-HRP (Medical Laboratories, Nagoya, Japan)

Normal goat serum (Medical Laboratories, Nagoya, Japan)

DifcoTM Skim milk (Becton, Dickinson and Company, Sparks, USA)

Rabbit anti-8-nitroguanine antibody (Made by Ma N. et al, Suzuka University of Medical Science, Japan)

Anti-8-hydorxy-2'-deoxyguanosine monoclonal antibody (Japan Institute for the Control of Aging, Shizuoka, Japan)

Rabbit peroxidase-anti-peroxidase (PAP) (ab28054, abcam, Tokyo, Japan)

Alexa 488-labeled goat antibody against mouse IgG (Molecular Probes Inc., Eugene, Oregon, USA)

Alexa 594-labeled goat antibody against rabbit IgG (Molecular Probes Inc., Eugene, Oregon, USA)

Other antibodies from abcam or Santa Cruz Biotechnology, Inc. USA.

Tris-HCl (Sigma, St. Louis, MO, USA)

Sodium chloride (Nacalai, Kyoto, Japan)

Sodium pentobarbital (Nacalai, Kyoto, Japan)

Ethanol (Nacalai, Kyoto, Japan)

204 Biomarker

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,

Rat, mouse, hamster, served as the donor tissue source in accordance with the guidelines Proper Care and Use of Laboratory Animals in Research. For the patient's operation samples, written informed consent was obtained from each patient, and the study protocol

**2.3.2 Chemicals and reagents for immunohistochemistry (IHC) and special equipment** 

3,3'-Diaminobenzidine tetrahydrochloride dehydrate (DAB) (Dojindo, Kumamoto, Japan)

Rabbit anti-8-nitroguanine antibody (Made by Ma N. et al, Suzuka University of Medical

Anti-8-hydorxy-2'-deoxyguanosine monoclonal antibody (Japan Institute for the Control of

Alexa 488-labeled goat antibody against mouse IgG (Molecular Probes Inc., Eugene, Oregon,

Alexa 594-labeled goat antibody against rabbit IgG (Molecular Probes Inc., Eugene, Oregon,

Dapi-Fluoromount-GTM Mounting Medium (Southern Biotech, Birmingham, USA)

guanosine; dG, deoxyguanosine and 8-BromoG, 8-bromoguanosine.

followed the ethical guidelines of the Declaration of Helsinki.

Malinol mounting medium (Muto Pure Chemicals Co. Tokyo,Japan)

Goat anti-rabbit -globulin-HRP (Medical Laboratories, Nagoya, Japan)

Rabbit peroxidase-anti-peroxidase (PAP) (ab28054, abcam, Tokyo, Japan)

Other antibodies from abcam or Santa Cruz Biotechnology, Inc. USA.

DifcoTM Skim milk (Becton, Dickinson and Company, Sparks, USA)

Normal goat serum (Medical Laboratories, Nagoya, Japan)

**2.3 Materials and animals 2.3.1 Donor tissue source** 

Silanized slides (Dako, Japan)

Science, Japan)

USA)

USA)

Aging, Shizuoka, Japan)

Tris-HCl (Sigma, St. Louis, MO, USA) Sodium chloride (Nacalai, Kyoto, Japan)

Sodium pentobarbital (Nacalai, Kyoto, Japan)

Xylene (Nacalai, Kyoto, Japan)

Paraformaldehyde (Nacalai, Kyoto, Japan)

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

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

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

Sliding microtome (RM2265, Leica, Germany)
