Atlas de histología vegetal y animal

Histological techniques


There are many fixatives described in the histological methods books. In this page, the most common fixatives used in histology labs will be mentioned. These have being proved as the most suitable for a better preservation of the tissue features. Chemical fixatives are the most frequently used, either as a single component or as solutions containing several fixatives.

Considering the mechanism of fixation, fixatives can be classified in two types: coagulant and cross-linking. Coagulant fixatives remove water from tissues leading to coagulation and denaturalization of proteins, mostly in the extracellular matrix. Cross-linking fixatives form chemical bonds between molecules of the tissue. Alcoholic fixatives are coagulants, such as Bouin and Carnoy, whereas formaldehyde and glutaraldehyde are cross-linking fixatives. Sometimes, a mix of the two types of fixatives is used.

Fixatives are also classified as additive and non-additive. Additive fixatives combine with molecules of the tissue so that the fixative, or some of its components, becomes part of the tissue and it is processed in the following steps of the histological protocol. They are mainly cross-linking fixatives and some coagulant fixatives. Non-additive fixatives, once performed the fixation, are remove from the tissue in later steps of the tissue processing. Alcohol and acetic acid are examples of non-additive fixatives.

Warning! Most of fixative solutions are toxic by inhalation or skin contact, some of them are carcinogenic. It is important to follow the safety guidelines for manipulating these substances.



Ethanol: CH3-CH2-OH

Ethanol, methanol and acetone. Their fixation mechanism is by dehydration and coagulation of proteins, mostly cytosolic proteins. The lipids are extracted from tissues, but carbohydrates are nor affected. Methanol is better fixative than ethanol because the tissue is not hardened and is better preserved. In general, they are good fixatives for small size samples, for preserving proteins, like enzymes, glycogen and some pigments. Blood smears and frozen tissue sections from unfixed samples are usually fixed with these fixatives. Since they do both dehydration and fixation at the same time, they can be used for preserving the samples. However, as a drawback, they normally produce hardening and retraction in the tissue, and lack mordant effect.

Acetic acid

Acetic acid: CH3COOH

Acetic acid. The effect of acetic acid on tissues is not a direct fixation, but a change of the colloidal state of proteins. Acetic acid is used at a concentration of 1 to 5 %. It is a very good fixative for nucleic acids and nucleoproteins. As drawbacks, acetic acid destroys mitochondria and does not fix well membranes and cytoplasm. It is commonly combined with other fixatives like Bouin (formaldehyde + acetic acid + picric acid) and FAA (formaldehyde + acetic acid + ethanol). In some fixative solutions is used to counteract the artifacts that may cause ethanol or picric acid..

Zinc chloride and zinc sulfate. As a fixative, zinc salts were used long ago, and later they became a component of several fixative solutions. Currently, they are combined with paraformaldehyde. They help paraformaldehyde with the fixation and preserves tissular antigens for immunohistochemical techniques by minimizing the antigen masking effect of paraformaldehyde. Mercury salts, which were used in the first fixative solutions, have been replaced by zinc salts. Fixatives containing zinc salts are not prepared in phosphate buffer, as it is common for other fixative substances. Furthermore, after fixation, zinc has to be removed from sample with thorough washes in distilled water.

Picric acid

Picric acid: C6H2OH(NO2)3

Picric acid. The fixation by picric acid is mediated by the coagulation of proteins produced by the picrate salts. Normally, 2 % to15% of a saturated solution of picric acid is combined with other fixatives. When the fixation time is set properly, picric acid is a good fixative for preserving the cellular structure, as well as glycogen and lipids. It has mordant effect, which is needed for some dyes of several post-embedding staining methods. It is a good practice to wash thoroughly the samples to remove the picric acid before paraffin embedding because picric acid makes difficult the infiltration of paraffin. Bouin is a widespread used fixative containing picric acid.

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Updated: 06-08-2018. 10:51