Formaldehyde fixation

Mason JT, O Leary TJ. Effects of formaldehyde fixation on protein secondary structure a calorimetric and infrared spectroscopic investigation. J. Histochem. Cytochem. 1991 39 225-229. 

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).

In 1991 Shi et al. published their seminal observation that high-temperature incubation of formalin-fixed, paraffin-embedded (FFPE) tissue sections in buffers for short periods led to improved immunohistochemical staining.1 However, more than 15 years later, heat-induced antigen retrieval (AR) remains largely an empirical procedure, requiring the optimization of several critical parameters by trial and error.2,3 Further improvements in AR will require an in-depth understanding of the chemistry of formaldehyde fixation and the molecular mechanism(s) underlying the AR method. 

Puchtler H, Meloan SN. On the chemistry of formaldehyde fixation and its effects on immunohistochemical reactions. Histochemistry 1985 82 201-204. 

Hua C, Langlet C, Buferne M, et al. Selective destruction by formaldehyde fixation of an H-2Kb serological determinant involving lysine 89 without loss of T-cell reactivity. Immunogenetics 1985 21 227-234. 

These functional characteristics reflect what is known about the chemistry of formaldehyde fixation, much of which has previously been reviewed by Fox and by Shi et al.1,2 Some significant facts about formaldehyde fixation of proteins include ... 

To these facts regarding formaldehyde fixation we can then enumerate some of the things that we know about antigen retrieval techniques ... 

Formaldehyde has several advantages over alcohols and acetone, particularly the superior preservation of morphological detail. When the specimen has to be embedded in paraffin or synthetic resin, formaldehyde fixation is the best choice. Formaldehyde is the simplest aldehyde. Its chemical formula is H2CO. It was first synthesized by the Russian chemist Aleksandr Butlerov in 1859. Discovered to be a tissue fixative originally by the German pathologist Ferdinand Blum in 1893, it... 

In most cases, fixation may be carried out at room temperature. Duration of formalin fixation depends on the nature and the size of the specimen, and may vary from 15 min to 24 h. Longer fixation may be associated with a partial loss of the antigenicity of the component of interest. After formalin fixation, tissue samples are washed in three changes of the buffered saline (PBS) from 15 min to 2 h, but not longer than 24 hours on the whole, since the formaldehyde fixation is partially reversible. After washing in PBS, specimens may be either snap-frozen in liquid nitrogen for subsequent cryosectioning, or dehydrated and embedded in paraffin or synthetic resin. 

Effect of Formaldehyde Fixation on Antigen-Antibody Binding... 

The exact mechanism by which HIER works is unknown. It is thought to reverse the masking effects of formaldehyde fixation and routine tissue processing. Hydrolytic-proteolytic cleavage of formaldehyde-related crosslinks, unfolding of inner epitopes, as well as the extraction of calcium ions from coordination complexes with proteins are among the hypothesized mechanisms (13-15). 

Formaldehyde works well as a primary fixative in this setting because it preserves cell morphology well and the time of exposnre to the fixative is short. Formaldehyde fixation at room temperatnre is very effective, bnt at 4°C, it is a very poor fixative. Longer fixation times (30-60 min) may be helpful for some antigens that are difficult to preserve. 

Colors and intensity of bands may differ after formaldehyde fixation compared with those obtained by glutaraldehyde fixation. Formaldehyde yields blackish and glutaraldehyde gives brownish bands. 

Formaldehyde introduces both intramolecular and intermolecular crosslinks between proteins involving hydroxymethylene bridges, which change the three-dimensional structure of proteins. Such changes involve the tertiary and quaternary structures of proteins, whereas the primary and secondary structures are little affected. It has been shown that the secondary structure of purified protein molecules remains mostly unaltered during fixation with formaldehyde (Mason and O Leary, 1991). Even when the quaternary structure is changed by formaldehyde fixation, the secondary structure can remain intact. 

Heat treatment, on the other hand, elevated cytoplasmic immunoreactivity of Bcl-2. However, nuclear and mitotic Bcl-2 immunoreactivity was clearly present when these cells were fixed with formaldehyde (3.6%), followed by postfixation with methanol for 10 min at -20°C. Treatment with ice-cold methanol makes the cell membrane permeable, allowing antibody access to intranuclear antigens without protein relocalization. Extensive protein crosslinking with formaldehyde is required for maintenance of intranuclear Bcl-2 immunoreactivity. In contrast to Bcl-2, Bax immunoreactivity was detected in nuclear and cytoplasmic compartments regardless of the duration of formaldehyde fixation used. 

On the other hand, available evidence supports the occurrence of the breakage of protein crosslinks, which allows the antibody access to the antigen. Conventional heat, dry or steam, breaks down reversible protein-protein, protein-nucleic acid, and protein-carbohydrate crosslinks introduced by formaldehyde and thereby unmasks the epitopes, as well as allowing the antibodies access to the epitopes. It is well known that most, if not all, crosslinks formed during formaldehyde fixation are destroyed upon heating even at 37°C... 

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