Immunohistochemistry (IHC) General protocol

Immunohistochemistry (IHC) General protocol




I. Required material
Reagents
- A primary antibody , labeled or not, against the target molecule
- Revelation kit
- Chromogen

Buffers
- Xylene
- Alcooholic baths at 70%, 80%and 95%
- Hydrogen peroxide (H2O2)
- Tris EDTA, Tris HCl
- Tween-20

Material
- Pipets and Pipet-aid
- Water bath


II. Experimentation length
- 10 mn of peroxydase blocking
- 30 mn of antigenic retrieval
- 1 h incubation of primary antibody
- 10 mn incubation of chromogen
- 2 h of rinsing steps and baths
- TOTAL : 4 hours


III. 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 5 minutes.
- 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 - 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 for 10 minutes.
- Stain in hematoxylinfor 5 minutes.
- Wash in water for 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.