Fixation with Formaldehyde

Hopwood D, Shdders W, Yeaman GR. Tissue fixation with phenol-formaldehyde for routine histopathology. Histochem. I 1989 21 228-234. 

Although fixation with formaldehyde has been a mainstay of tissue preservation for well over 100 years, the chemical basis for this effect has been elucidated very slowly over that time. Exposing tissue to formaldehyde rather rapidly stops the enzymatic degradation and bacterial growth that begin immediately after tissue is excised or an organism dies, and causes the tissue to shrink and become hard relative to the unfixed state. This results in a tissue... 

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. 

Formaldehyde prevents the extraction of glycogen but does not preserve soluble polysaccharides. Acid mucopolysaccharides are also not preserved unless they are bound to proteins (3). Formaldehyde is a good fixative for lipids, particularly if 1-2 mM Ca " or Mg + are included in the fixative vehicle (4,5,11). Membrane fixation is improved by reducing lipid extraction (4). It is also thought that fixation with formaldehyde lowers the solubility of membrane phospholipids in water (11). 

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). 

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. 

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). 

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. 

The effect of prolonged fixation with formaldehyde on the antigenicity of the nucleus may differ from that of the cytoplasm. This phenomenon is exemplified by Bcl-2 and Bax, members of the same family of proteins involved in apoptosis regulation these proteins reside in the cytoplasm as well as in the nucleus. It was recently demonstrated that prolonged fixation with formaldehyde alone irreversibly reduced nuclear or mitotic Bcl-2 immunoreactivity even after heat-mediated antigen retrieval in monolayers of MCF-7 human breast cancer cells (Hoetelmans et al., 2001). 

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. 

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