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 microwave (850W) for 10 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 breast cancer tissue (A, 4 μm), human stomach adenocarcinoma (B, 4 μm), and human colon adenocarcinoma (C, 4 μm), stained with anti-Cytokeratin 8 antibody (DB 098) shows strong positive cytoplasmic immunostaining of neoplastic cells (A), strong positive cytoplasmic immunostaining of tumor cells (B, C).