Permeabilization for IHC
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Permeabilization is required when the antibody needs access to the inside of cells in order to detect the target antigen.
Such antigens include intracellular proteins and cytoplasmic epitopes of transmembrane proteins. Solvents or detergents are typically used for permeabilization.
Solvents can be used after fixation with crosslinking agent such as formaldehyde. Recommended for cytoskeletal, viral and some enzyme antigens.
Detergents can either be harsh (eg Triton™ X-100 or NP-40) to disrupt proteins, or mild (eg Tween 20®, saponin or digitonin) so as to not dissolve plasma membranes. Detergent permeabilization can significantly improve antibody access to antigens in the cytoplasm, on the cytoplasmic face of the plasma membrane, and soluble nuclear antigens.
Solvent and detergent guidelines
| Solvents | Comments | |
| Solvents | Acetone
Methanol | Acetone fixation will also permeabilize Methanol fixation can be used to permeabilize but is not always effective |
| Detergents | Triton™ X-100 or NP-40 | Use 0.1 to 0.2% in PBS for 10 min only |
| Tween 20®, saponin, digitonin and Leucoperm | Use 0.2 to 0.5% for 10 to 30 min |
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Tween 20® is a registered trademark of Croda Americas. Triton is a trademark of the Dow Chemical Company.