No single fixative is appropriate for every cell line or target protein, so optimal conditions should be determined empirically.
Two general classes of fixative can be used:
- Cross-linking reagents
- Cross-linkers (usually aldehydes, such as formaldehyde) form a network of intermolecular bridges by linking antigens through free reactive groups, primarily amino groups.
- Cross-linking helps to preserve cell structure.
- A separate step is usually required to permeabilize the cell membrane.
- Cross-linking reagents may denature some proteins and reduce their antigenicity.
- Organic solvents
- Organic solvents dehydrate the cell. This precipitates cellular proteins and causes cells to adhere to the plastic surface of the microplate.
- A separate permeabilization step may not be needed.
- Organics may denature or damage some proteins, and severely reduce their antigenicity.