Cell and Tissue Fixation

To ensure that the antibody is free access to its antigen, the cells must be fixed and permeabilized. In general, fixation strengths and times for cells are much shorter than those of thicker, structurally complex tissue sections. For immunocytochemistry, sample preparation is primarily to fix the target cells to the glass slides. Perfect fixation would immobilize the antigens, while retaining authentic cellular and subcellular structure and allowing unhindered access of antibodies to all cells and subcellular compartments.

The fixation methods are generally classified into two classes of organic solvents and cross-linking reagents. Organic solvents, such as alcohols and acetone, remove lipids and dehydrate the cells, while precipitating proteins on the cellular structures. Cross-linking reagents, such as paraformaldehyde, typically form intermolecular bridges through free amino groups to create a network of linked antigens. Cross-linking reagents protect cell structure better than organic solvents, but may reduce the antigenicity of certain cell components and require an additional permeabilization step to allow access of the antibody to the specimen. Fixation with both methods can denature protein antigens, and for this reason, antibodies prepared against denatured proteins may be more useful for cell staining.

Different fixation methods are described here. Each method has advantages and disadvantages, and the choice of which method depends on the nature of the antigen being examined and on the properties of the antibody used.

Table 1. Detailed fixation and permeabilization methods for cell culture samples.

Table 2. Fixation and permeabilization methods for tissue samples.

For research use only. Not for any other purpose.