Protein-formaldehyde reactions tissue

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. 

Hydrazide-based cyanine dyes are reactive with common formaldehyde fixatives for cell and tissue studies. This enables these dyes to function as general stains for protein-rich areas within cells, and they get crosslinked into place by the formaldehyde reaction process. 

Rapid and uniform fixation throughout the tissue block with formaldehyde can be obtained at high temperatures, for example, in a microwave oven. Such temperatures enhance the speed and extent of formaldehyde reaction with proteins by dissociating the methylene glycol to formaldehyde as well as by depolymerizing the oligomers of methylene glycol (Boon et al., 1988). 

These initial findings do not exclude other possible formaldehyde-induced reactions with tissue proteins. Notably, this first model system was not designed to detect the role of lysine residues. Lysine has a propensity to react with and form a variety of different types of cross-links with other amino acids in the presence of formaldehyde.1,3 417 Therefore, it is likely to also be important in reactions with formaldehyde. In fact, peptides with internal lysine residues were purposefully excluded from this initial study for technical reasons. To explore the importance of lysine residues in antigen retrieval, an alternative method was employed. 

A simpler procedure consisting of protein precipitation using MeCN and TCA following formaldehyde derivatization was used for the determination of AMP in milk samples. The use of MeCN and TCA described in this method resulted in a clear supernatant and the highest recoveries (93% with CV of 6.1%). The derivatization reaction was done at 100°C for 30 min. The fluorescence of AMP derivatives was at least 20 times higher than that of AMO derivative thus, no preconcentration step was needed, resulting in the simplicity of this assay. A coextracted interfering unknown compound was observed at or near the retention time of the AMP derivative. This did not affect the accuracy (less than 10%) of the determination (74). A similar procedure was reported for beef, pork, chicken, and catfish tissue samples (75). 

Figure 12.4 In the presence of formaldehyde, the exocyclic nitrogen (encircled, upper left) on a nucleotide forms an Af-hydroxymethyl adduct (upper center). Further denaturing reactions of formalin-fixed nucleic acids during tissue processing lead to a compound ethoxymethyl adduct (lower right), fragments from depurination (lower left), cross-hnks to associated proteins (not shown), and hydrolysis of phosphodiester bonds (not shown).

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