Antigen retrieval formalin fixation, peptide

In order to model the effect of formalin fixation and antigen retrieval on antibody immunoreactivity, we used a peptide epitope array (Fig. 16.5). The peptide epitopes are derived from the exact sequences in the native proteins. In this experiment, the initial findings are similar to those shown in Figure 16.1, but they are then extended by allowing for the role of adjacent proteins. 

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.

A reasonable objection to any in vitro model is whether it accurately mirrors the actual process. A strength of this model is that the peptides in the array, mounted on the microscope glass slide, are the very same as the antibody epitopes in the native proteins. Therefore, the types of formaldehyde-induced chemical reactions at or near the epitope are the same as would likely occur in a tissue sample. An additional strength of the model is that the experimental data using the peptide array completely account for the loss of immunoreactivity after formalin fixation and the recovery of immunoreactivity after antigen retrieval (Fig. 16.5). Nonetheless, our data do not prove that the model accurately represents formaldehyde reactions in tissue specimens. For example, our data do not exclude other causes of steric interference. 

Even among the group of peptide epitopes, there was a striking variability in the speed of formalin fixation and antigen retrieval, suggesting hetero-... 

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