Home > Protocols > Immunoshistochemical detection of 4-HNE Rev.080612
Immunohistochemical detection of 4-hydroxy-2-nonenal (4-HNE) by HNEJ-2 antibody.
This is an application example for anti 4-HNE monoclonal antibody (clone HNEJ-2). Experimental conditions should be settled by each user.
Fixation: Fixation by Bouin's Solution is recommended. Antigen retrieval process is not needed. But if tissue samples or cultured cells are fixed by formalin, staining may be improved by antigen retrieval.

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.

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. Please refer technical information.
Anti 4-HNE antibody: Apply HNEJ2 antibody (10-20 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).
oxidative sress markers/oxidative stress test
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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oxidative sress markers/oxidative stress test

oxidative sress markers/oxidative stress test
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