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 at 95ºC in water bath for 30 minutes.
- 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 tonsil tissue (A, 4 μm), human colon adenocarcinoma (B, 4 μm), and human liver tissue (C, 4 μm), stained with anti-Cytokeratin 19 antibody (DB 103) shows strong positive cytoplasmic immunostaining of the squamous epithelial cells (A), strong positive cytoplasmic immunostaining of the neoplastic cells (B), and positive cytoplasmic immunostaining of the bile duct epithelial cells (C).