Fixation and Fixatives (3) – Fixing Agents Other than the Common Aldehydes: Leica Biosystems
Glyoxal is the major ingredient in several proprietary fixatives particularly applicable to . Effects of fixation on immunohistochemistry, reversibility of fixation and. This working surgical pathologist would like to add - and solicit - some comments on Histonet. "Glyoxal Fixation and Its Relationship to Immunohistochemistry". Glyoxal fixation and its relationship to immunohistochemistry. Dapson, R., W.; Feldman, A., T.; Wolfe, D. Journal of Histotechnology 29(2): Glyoxal is.
In addition, solutions of heavy metal salts — mercury, chromium or osmium Zenker's fixative, chromic acid and potassium dichromate, osmium tetroxide are used for fixation. Some of these fixatives have been tested for preparation of sample for immunohistochemical staining. For example, it was noted that after fixation in the Carnoy's solution, many antigens can be well detected, even better than after the standard fixation in neutral formalin .
Fixation with methacarn also allowed for visualization of certain epitopes better than after fixation with formalin . Thus, different antigens were identifiable better or worse after the treatment with different fixative reagents; according to some authors [71, 72], after fixation with neutral formalin most of the sought antigens could be visualized upon immunohistochemical staining.
However, other studies [60, 73] showed poor detectability of antigens after fixation in formalin as compared with other tested fixatives: The detection of epitopes after prolonged fixation in neutral formalin was particularly poor [7, 74, 75]. The PLP fixative was shown to preserve the intact antigenic structure much better than formalin; for a number of proteins fixed with PLP, there is no need in the subsequent antigens unmasking .
However, when the biological sample preservation was compared between different fixative agents, PLP was found to cause the strongest compression of tissues upon fixation.
In this respect, formalin was less aggressive, and the zinc-containing fixatives were the least compressive fixatives . There is a report that the antigens of invertebrate tissue remained well-preserved after fixation in a mixture of uranyl acetate, trehalose, and methanol .
Regarding the toxicity of heavy metals, there were attempts to avoid their use for fixation. As it was reported, mercury chloride could be replaced with zinc salts chloride, sulfate so the resulting reagent contained zinc ions as the only fixing agent .
Although the mechanism of fixation by zinc ions is not fully understood, biological samples fixed with zinc showed a good preservation quality of cells and extracellular material; notably, the zinc-containing fixative was advantageous for the preservation of antigens in comparison with neutral formalin  or periodate-lysine-paraformaldehyde .
Nevertheless, as noted by the authors, aqueous solutions of zinc salts penetrate the depth of a fixed sample not fast enough, which can cause an uneven immunoreactivity of antigens located on the surface or in deep layers of the tissue.
Immunohistochemical study of nervous tissue definitive and embryonic and peripheral organs fixed in zinc-ethanol-formaldehyde allowed for detecting a large number of proteins: In addition, immunohistochemical visualization of some antigens did not require heat induced epitope retrieval. In a search for fixatives that would ensure good preservation of every morphological detail and minimally change the antigens, commercial companies offer new types of fixing solutions for immunohistochemical analysis.
These trade names often conceal combinations of well-known aldehyde or alcohol fixatives, sometimes added with original admixtures. For example, the UPM fixative is a mixture of ethanol, methanol, 2-propanol, and formalin; CyMol — ethanol, methanol, and 2-propanol; Greenfix — ethanol and ethanediol.
However, the precise composition of these fixatives is not disclosed to the public; therefore, it is difficult to assess the effects of individual components of a commercial product on the preservation and antigenic properties of biological samples. Fixation of biological material for electron microscopic immunocytochemistry requires special conditions. Usually the fixation is made with glutaraldehyde, osmium tetroxide or uranyl acetate; the latter two are also used for contrasting the sample, which is needed for viewing it by means of an electron microscope.
For a combined light and electron microscopy immunocytochemical examination, a mixture of glutaraldehyde and paraform — the so-called Karnovsky's fixative and its variants — is used [10, ]. Acrolein acrylic acid aldehyde is also used thanks to its relatively mild masking impact on the tissue antigens; however, acrolein is extremely toxic, therefore it is used rarely or in a combination with glutaraldehyde or formaldehyde .
Osmium tetroxide has been shown to mask antigens [10,], which makes its use in immunocytochemistry problematic. Due to this, tannic acid has been suggested to replace osmium; tannic acid produces a well-contrasted material for electron microscopy and also provides for the detection of antigens at the ultrastructural level . Another option is to use glutaraldehyde alone and dissolve it in a buffer with a balanced ionic composition .
Good preservation of the ultrastructure and the antigens was achieved after fixing the material in periodate-lysine-paraformaldehyde . Conclusion According to the literature, over the recent 25 years of immunohistochemical studies, important improvements in the techniques of biological fixation have been achieved. Yet, none of the fixatives known to date has an ideal combination of properties that enables to obtain high-quality histological preparations and that does not interfere with identification of any antigens in the subsequent immunostaining.
The only conclusion from this review is that the optimal fixation protocol should be selected or developed for any specific protein or antibody, considering the experience of others with related proteins. It is not advisable to focus on commercial fixatives with unknown compositions, since their production may be stopped one day for the commercial or technical reasons, and the researcher may be unable to accurately reproduce the conditions required for successful fixation.
The work was funded by the State-sponsored grant No. There are no conflicts of interests related to this study. Fiksatsiya materiala dlya gistologicheskogo issledovaniya. Saint Petersburg; ; p. Formaldehyde, formalin, paraformaldehyde and glutaraldehyde: Micros Today ; 8 Macromolecular changes caused by formalin fixation and antigen retrieval. Biotech Histochem ; 82 3: Kinetic studies of formaldehyde binding in tissue. Biotech Histochem ; 69 3: Cell fixatives for immunostaining. Methods Mol Biol ; The effects of progressive formaldehyde fixation on the preservation of tissue antigens.
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Although acrolein penetrates tissue rapidly and is extremely reactive it has not found widespread acceptance because it is unpleasant to use a powerful lacrimatory agentunstable at alkaline pH, and readily forms polymers. It has mainly been used for enzyme histochemistry and for the fixation of plant material. Following fixation residual carbonyl groups in the tissue will cause background staining with techniques such as PAS.
CHO is a bifunctional aldehyde that reacts in a similar manner to formaldehyde producing a similar morphological picture. Each of the aldehyde groups is potentially reactive and cross-links can be formed. Glyoxal is the major ingredient in several proprietary fixatives particularly applicable to microwave fixation as well as for routine work.
Glyoxal fixation: how it works and why it only occasionally needs antigen retrieval.
Although it is an irritant and potentially sensitizing on skin contact it is not currently classified as a carcinogen, although warnings are provided as to its potential mutagenic effects. It reacts with DNA forming adducts in an unstable and reversible manner. It is said to be biodegradable so that disposal will be easier than for formalin.
Because of its volatility it must be handled with great care under a fume hood because it will readily fix the conjunctiva of the eye and the nasal mucosa. The most important fixation reactions of osmium tetroxide are those involving unsaturated bonds of lipids and phospholipids as it is one of the few fixatives that stabilises lipids.
During fixation osmium tetroxide is reduced to lower oxides which are black and insoluble and these are deposited in tissues, particularly on membranes. Because osmium is a heavy metal it scatters electrons and thus adds electron density to the electron microscope image.
The myelin sheaths are preserved and stained black by this process.
In this example a paraffin section has been prepared but excellent results can be achieved when polymerizing resins epoxies or methacrylates are used instead. The reactions can be enhanced or selectively blocked by choosing a particular type of carbodiimide, pH, temperature, or catalyst. Because peptide bonds may form as one result of the fixation reactions and subsequently these may be selectively broken using proteases, this group of compounds is thought to have some potential for use in immunohistochemistry as well as for routine histology.
Carbodiimides are already used for the preparation of immunogens.
Glyoxal fixation and its relationship to immunohistochemistry
Chloro-s-triazides cyanuric chloride has been used for salivary gland mucins and immunofluorescence. Diisocyanates have been used to attach fluorescent tags to proteins, while Diethylpyrocarbonate DPC reacts with tryptophan residues and has been used as a vapour-phase fixative for freeze-dried tissue.
In an appropriate buffer solution it has been proposed as a fixative for small specimens.
Maleimides appear to form some cross-links with proteins and Benzoquinone reacts with amines, amino acids and peptides and has been used to fix peptides in endocrine tissues for immunohistochemistr. Although the mechanisms by which it fixes tissue are not fully understood it is known to react with amines, amides, amino acids and sulphydryl groups, the latter being prominent in its reaction with cysteine where it is thought to produce cross-links.
It is a powerful protein coagulant which leaves tissue in a state which produces strong staining with acid dyes. It reacts with phosphate residues of nucleic acids and effectively fixes nucleoproteins. There are several disadvantages to using fixatives containing mercuric chloride. Apart from the corrosive nature of mercuric chloride, mercury is highly toxic, can be absorbed through the skin and is a cumulative poison.
In most countries there are strict rules about disposal of mercury and compounds containing mercury. During fixation with fixatives containing mercuric chloride a crystalline or amorphous greenish-brown artefact pigment of mercury is randomly deposited in tissues. A subsequent treatment with sodium thiosulphate then removes residual iodine. Mercuric chloride-based fixatives tend to penetrate poorly and if fixation is prolonged tissues become very hard and are prone to shrinkage during processing.
Zinc chloride and zinc sulphate have been accepted fairly widely as being suitable and there are now many proprietary B-5 substitutes available. A section of kidney that contains randomly distributed greenish-brown deposits, an artefact produced when mercuric chloride is a component of the fixative used.
In this case the section was not treated to remove the deposit prior to staining. Zinc salts Zinc sulphate ZnSO4 and zinc chloride ZnCl2 are used as substitutes for mercuric chloride in a number of formulated and proprietary fixatives, the sulphate being more popular because it is potentially less corrosive than the chloride which has been reported as causing problems in tissue processors 6 see Part 4.
Zinc salts will react with a range of tissue end groups including amino, carboxyl and sulphydryl, forming reversible reaction products some of which can be removed with a citrate or EDTA wash. Zinc is said to enhance fixation and staining, particularly of nuclei, in a similar way to mercuric chloride.