Home > Protocols > Immunoshistochemical detection of 8-OHdG/8-oxo-dG Rev.080611
Immunohistochemical detection of 8-hydroxy-2'-deoxyguanosine (8-OHdG/8-oxo-dG) by N45.1 antibody.
This is an application example for anti 8-OHdG monoclonal antibody (clone N45.1). Experimental conditions should be settled by each user.
Fixation: For formalin-fixed tissue and cultured cells, antigen retrieval process may be useful. Tissue samples are fixed by Bouin”Ēs Solution, antigen retrieval process will not be needed.

1) Fix the fresh tissues with Bouin”Ēs Solution (Saturated Picric Acid: Formaldehyde: Acetic Acid : H2O = 15:5:1:10) overnight. Please note that use of specimens fixed in the same conditions are recommended for quantitative analyses.

2) Deparaffinization (In case of paraffin embedded specimens).Deparaffin with xylene and ethanol.

3) Antigen Retrieval (In case of fixation with formaldehyde).Microwave method or autoclave method is recommended.
Blocking: Apply normal rabbit serum (Diluted to 1:75) (DAKO corporation, Kyoto, Japan) to the specimens for the inhibition of non-specific binding of secondary antibody.

For blocking reagent, please use normal serum from the same species as second antibody used. For example, if you use biotin-labeled goat anti mouse IgG, normal goat serum may be suitable.
If sample tissues are from mouse, blocking of internal mouse IgG will be needed. See technical information.
Anti 8-OHdG antibody: Apply N45.1 (1-10 µg/mL) to the specimens, incubate overnight at 4 °C.
Secondary antibody: Apply biotin-labeled rabbit anti-mouse IgG serum (Dako; Diluted to 1:300) for 40min at room temperature.
ABC method: Apply avidin-biotin-alkaline phosphatase complex (Vector Lab., Burlingame, CA; Diluted to 1:100) for 40min at room temperature.
Staining: Use a kit of black substrate for alkaline phosphatase (BCIP/NBT, Vector).
Antigen retrieval: By using a microwave oven
1) Put sample slides into 500mL glass beaker with 10mM citrate buffer pH6.0 and heat. Please take care that samples are not detached from slide glass.
2) Keep boiling for 5 minutes.
3) Cool down slowly in 1 hour.
If you are using autoclave method, heating condition should be 10 minutes at 121 °C.

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