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Fixation Conditions


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.

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