Fixation in Formaldehyde

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... 

Fixation by formaldehyde virtually eliminates enzymatic activity in a process that is sometimes reversible (vide infra). Dehydration and embedding significantly reduces the reversibility of this process, suggesting that dehydration and embedding facilitate additional chemical reactions that are not observed in aqueous solution. 

Citraconic anhydride also has been used to reverse the effects of formalin fixation in tissue sections. Namimatsu et al. (2005) found that heating deparafinized tissue sections in a dilute solution of citraconic anhydride broke the formaldehyde crosslinks and restored antigen recognition of proteins within the samples. 

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 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). 

Fixation Fixation is the process by which the protein of cells is denatured, or cross-linked, and preserved. Fixation in flow cytometry is used to inactivate hazardous biological material and also to preserve stained cells when there is not immediate access to a flow cytometer. Fixation is also important in preserving proteins before detergent permeabilization for intracellular staining. Formaldehyde is often the fixative of choice for flow cytometry because it preserves the forward and side scatter characteristics of cells (but does cause some increase in their autofluorescence). 

It is also likely that certain antigens have optimal durations of fixation. It has been shown that for the immunohistological demonstration of estrogen receptor, a minimum fixation time of 6-8 hrs in formalin is required for consistent results (Goldstein et al 2003). This minimum time is particularly critical in small biopsies and needle cores which tend to receive insufficient or weak initial formaldehyde fixation followed by extraction/fixation in ethanols used for dehydration in the tissue processor, resulting in inconsistent demonstration of some tissue antigens. 

In the TIJNEL test, it is necessary to permeabilize the cells to introduce the enzyme and the deoxynucleotides, but the permeabilization is carried out after weak fixation with formaldehyde, so that low molar mass fragments are not lost. Permeabilization is carried out in an ice bath, followed by labeling with the reaction solution. 

The animals are sacrificed and the thoracic aorta is removed, cleaned of surrounding tissues, and longitudinally cut and opened for fixation with formaldehyde. The tissue is stained with oil red. The percentage of the intimal surface covered by the oil red positive lesions is calculated with a computerized planimeter. In animals fed a normal diet, the aorta does not show any staining, whereas in cholesterol-fed rabbits the aorta shows severe atherogenic lesions. 

---