Home > Protocols > Immunoshistochemical detection of HEL Rev.080611
Imminohistochemical detection of hexanoyl-Lys adduct (HEL) by monoclonal antibody.  
This is an application example for anti hexanoyl-Lys adduct (HEL) monoclonal antibody. Experimental conditions should be settled by each user.
Fixation: Formalin fixation. Slides should be prepared 3 µm thick.
Antigen retrieval is not needed, but in some cases antigen retrieval may improve staining.
Blocking: Apply normal rabbit serum (DAKO corporation, Kyoto, Japan) in 1% BSA/PBS for 30 minites 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 HEL antibody: Apply anti HEL antibody (2 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 (optional):
1) Put sample slides into 500mL glass beaker with 10mM citrate buffer pH6.0 and heat by microwave oven. Please take care that samples are not detached from slide glass.
2) Keep boiling for 5 minutes.
3) Cool down slowly in 1 hour.
Antibody product Instruction manual (PDF) Technical information

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