Theoretic consideration of fixation mechanism

Aldehyde crosslinks are formed between protein molecules, in particular with the basic amino acid – lysine. Only those lysine residues on the exterior of the protein molecule react with aldehydes. Because protein is a universal constituent of the cell, present not only in the cytosol but also in the cytoskeleton and the plasma membrane, soluble proteins can be cross-linked by aldehydes to each other and to the proteins throughout the cell. Crosslinks are made primarily on the hydrophilic areas of proteins. The reaction between aldehyde and protein is pH-dependent, progressing more rapidly at higher pH values. The reaction is reversible over the first 24hr of reaction time. Crosslinking denatures proteins to some extent, especially at higher concentrations. Notably, crosslinked proteins in the cell may form an interlocking meshwork, which could either restrict antibody access or trap antibodies in the meshwork, causing fixation artifacts.

Dehydrants such as alcohols and acetone remove and replace free water in the cell and cause a change in the tertiary structure of proteins by destabilizing the hydrophobic interaction and the hydrogen bonds. Hydrophobic areas, frequently found on the inside of protein molecules, are exposed due to the repulsion of water, allowing antibody access to otherwise inaccessible protein domains. Hydrophilic areas on the outside of protein molecules, which are loosely bound by hydrogen bonds, are destabilized by the removal of water. The conformational changes in the protein molecules may cause water soluble proteins to become insoluble, a change that will introduce fixation artifacts by altering protein localization.