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| Cat. #: |
DB 100-1 |
| Volume: |
1 ml |
| Dilution: |
1:100 - 200 |
| Application: |
IHC-P, IHC-Fr |
| Availability: |
Available immediately |
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Protocols in PDF format to download
IHC-P protocol
- Deparaffinize the section in 3 changes of xylene, 5 minutes each.
- Wash the section in 96%, 80% and 70% benzyl alcohol for 5 minutes each.
- Rinse in distilled water.
- Block the endogenous peroxidase by incubating the tissue in 3% hydrogen peroxide (H2O2) for 10 minutes.
- Wash in distilled water.
- For antigen retrieval: immerse the slide in Tris-EDTA buffer, pH 9.0, 0.05% Tween-20* and incubate in water bath at 95˚C for 30 minutes. (Alternatively adjust to your own protocol, keeping the required pH)
- Remove the staining to room temperature and let the slide to cool (in TRIS-EDTA buffer, pH 9.0) for 15 minutes.
- Rinse in distilled water.
- Wash in 0.05 M Tris-HCl , pH 7.6 buffer supplemented with 0.2% of Tween-20 (buffer A) for 5 minutes.
- For concentrated products: Incubate the section with primary antibody diluted in buffer A at the dilution 1:100- 1:200 for 1 hour in the closed wet chamber.
For RTU products: Incubate the section with primary antibody(ready to use) for 1 hour in a closed wet chamber.
- Wash twice 5 minutes with buffer A.
- Apply the secondary antibody (the protocol depends on the supplier), and proceed to standard immunohistochemistry protocol (HRP – Peroxide – DAB).
- Wash twice 5 minutes with buffer A.
- Apply the chromogen (DAB), 10 minutes.
- Wash in water – 10 minutes.
- Stain in hematoxylin for 5 minutes.
- Wash in water – 10 minutes.
- Dehydrate the section in 2 changes of 96% benzyl alcohol for 5 minutes each.
- Wash the section in 2 changes of xylene for 2 minutes each.
- Mount the slide for observation.
Formalin-fixed and paraffin-embedded human squamous cell skin carcinoma tissue (4 μm) stained with anti-Cytokeratin 16 antibody (DB 100) shows strong positive cytoplasmic immunostaining of neoplastic cells.