Fixing Attached Cells for Staining

For cell staining, fixation methods decrease generally into two classes, organic solvents and cross-linking reagents. Organic solvents such as alcohols and acetone remove lipids and dehydrate the cells, 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. Choosing between fixation in organic solvents or cross-linking agents is empirical. There are no general rules to decide between the two and both procedures are described here. Both methods may denature protein antigens, and for this reason, antibodies prepared against denatured proteins may be more useful for cell staining. In some instances, anti-denatured-protein antibodies are the only ones that can work. Fixation in protein cross-linking reagents such as paraformaldehyde or glutaraldehyde preserves cell structure better than organic solvents but may reduce the antigenicity of some cell components. Simple fixation with paraformaldehyde or glutaraldehyde does not allow the antibody to access the specimen and therefore is followed by a permeabilization step using an organic solvent or nonionic detergent. Using the organic solvent is easy, but it can destroy certain elements of the cell architecture, although prior fixation with paraformaldehyde does help to preserve the cellular structure. If preservation of cell structure is important, the best first choice would be to use a nonionic detergent.