| Home > Protocols > Immunoshistochemical detection of HEL |
Rev.080611 |
| 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.
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| 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). |
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| 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. |
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