To ensure free access of the antibody to its antigen, the cells must be fixed and permeabilized. In general, fixation strengths and times are considerably shorter for cells than on the thicker, structurally complex tissue sections. For immunocytochemistry, sample preparation essentially entails fixing the target cells to the slide. Perfect fixation would immobilize the antigens, while retaining authentic cellular and subcellular architecture and permitting unhindered access of antibodies to all cells and subcellular compartments. Wide ranges of fixatives are commonly used, and the correct choice of method will depend on the nature of the antigen being examined and on the properties of the antibody used. Fixation methods fall generally into two classes: organic solvents and cross-linking reagents. Organic solvents such as alcohols and acetone remove lipids and dehydrate the cells, while precipitating the proteins on the cellular architecture. Cross-linking reagents (such as paraformaldehyde) form intermolecular bridges, normally through free amino groups, thus creating a network of linked antigens. Cross-linkers preserve cell structure better than organic solvents, but may reduce the antigenicity of some cell components, and require the addition of a permeabilization step, to allow access of the antibody to the specimen. Fixation with both methods may denature protein antigens, and for this reason, antibodies prepared against denatured proteins may be more useful for cell staining. Different fixation methods are described. The appropriate fixation method should be chosen according to the relevant application.

1.   Acetone Fixation

• Fix cells in -20°C acetone for 5-10 minutes.

• No permeabilization step needed following acetone fixation.

2.   Methanol Fixation

• Fix cells in -20°C methanol for 5-10 minutes.

• No permeabilization step needed following methanol fixation.

3.   Ethanol Fixation

• Fix cells in cooled 95% ethanol, 5% glacial acetic acid for 5-10 minutes.

4.   Methanol-Acetone Fixation

• Fix in cooled methanol, 10 minutes at –20 °C.

• Remove excess methanol.

• Permeabilize with cooled acetone for 1 minute at –20 °C.

5.   Methanol-Acetone Mix Fixation

• 1:1 methanol and acetone mixture.

• Make the mixture fresh and fix cells at -20 C for 5-10 minutes.

6.   Methanol-Ethanol Mix Fixation

• 1:1 methanol and ethanol mixture.

• Make the mixture fresh and fix cells at -20 C for 5-10 minutes.

7.   Formalin Fixation

• Fix cells in 10% neutral buffered formalin for 5-10 minutes.

• Rinse briefly with PBS.

• Permeabilize with 0.5% Triton X-100 for 10 minutes.

8.   Paraformaldehyde-Triton Fixation

• Fix in 3-4% paraformaldehyde for 10-20 minutes.

• Rinse briefly with PBS.

• Permeabilize with 0.5% Triton X-100 for 10 minutes.

9.   Paraformaldehyde-Methanol Fixation

• Fix in 4% paraformaldehyde for 10-20 minutes.

• Rinse briefly with PBS.

• Permeabilize with cooled methanol for 5-10 minutes at –20 °C.