Cross-linking fixation

Cross-linking fixation - reactive groups bind proteins and lipids in cells and holds them in the same position as they were in living cell the fixative of choice for immunocytochemistry. 

Rait VK, Xu L, O Leary TJ, et al. Modeling formalin fixation and antigen retrieval with bovine pancreatic RBase A II. Interrelationship of cross-linking, immuno-reactivity, and heat treatment. Lab. Invest. 2004 84 300-306. 

Finally temperature and tissue type do have an effect on the penetration and fixation. This, combined with a failure to appreciate protein cross-linking time or actual fixation time required for formaldehyde, is probably the main reason for intra- and interlaboratory variation. 

During the tissue fixation process, proteins are cross-linked, causing some epitopes to become undetectable by the staining protocols.10 HIAR reverses this effect, allowing these epitopes to be stained, and therefore has become increasingly important for many IHC staining protocols.19-22 However, the available automated IHC platforms vary in their ability to perform online HIAR. 

Figure 12.3 Hydroxymethyl adducts from formaldehyde fixation form highly reactive imines in the presence of ethanol during tissue processing, giving off a molecule of water (upper). While still in alcohol, imines may either form more complex ethoxymethyl adducts or will cross-link to neighboring reactive groups (lower).

Fowler CB, O Leary TJ, Mason JT. Modeling formalin fixation and histological processing with bovine ribonuclease A effects of ethanol dehydration on reversal of formaldehyde-induced cross-links. Lab. Invest. 2008 88 785-791. 

Figure 16.5 Immunostained peptide arrays after various treatments of fixation, protein cross-linking, and antigen retrieval, as indicated at the top. Each row has a different peptide that is immunoreactive for the antibody denoted to the left. Column A represents the baseline condition, without any treatment whatsoever. Column B shows immunoreactivity of each peptide after overnight formalin fixation. Column C shows the immunoreactivity after first coating the array with an irrelevant protein (casein) followed by overnight formalin fixation. Column D illustrates the immunoreactivity of the peptides after the treatment of column C, and then antigen retrieval. Reproduced with permission from Reference 15, 2006 American Society for Clinical Pathology.

These data suggest that the loss of immunoreactivity after formalin fixation involves a protein cross-linking reaction. The first amino acid is at or near the antibody epitope. The second can be on the same protein or on another nearby protein. A variety of different chemical reactions with formaldehyde can be occurring. The common theme is that regardless of the details of the formaldehyde-induced cross-linking reaction, steric interference prevents antibodies from gaining access to the epitope. 

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