Tissue Fixation and Fixatives

The importance of fixation

In order to study tissues with a microscope they must be preserved (fixed) and cut into sections thin enough to be translucent. Fixation is a critical step in the preparation of histological sections. If it is not carried out under optimal conditions or if fixation is delayed, a tissue specimen can be irreversibly damaged. No matter how much care is subsequently taken in tissue processing, microtomy and staining, the morphological and histochemical information obtainable from the specimen will be compromised.

The broad objective of tissue fixation is to preserve cells and tissue components in a “life-like state” or as little alteration as possible to the living tissue, and to do this in such a way as to allow for the preparation of thin, stained sections. The choice of fixative and fixation protocol may depend on the additional processing steps and final analyses that are planned. There is no perfect fixative, though formaldehyde comes the closest. Therefore, a variety of fixatives are available for use, depending on the type of tissue present and features to be demonstrated.

 

Type of fixation

Fixation of tissues can be achieved by chemical or physical means.

Physical methods include heating, micro-waving and cryo-preservation (freeze drying).

Chemical fixation is usually achieved by immersing the specimen in the fixative (immersion fixation) or, in the case of small animals or some whole organs such as a lung, by perfusing the vascular system with fixative (perfusion fixation). For some specialised histochemical procedures fixatives have occasionally been applied in the vapour form. For example paraformaldehyde and osmium tetroxide can be used to vapour-fix freeze-dried tissues.

 

Mechanism of Fixation

The two main mechanisms of chemical fixation are cross-linking and coagulation.

Cross-linking involves covalent bond formation both within proteins and between them, which causes tissue to stiffen and therefore resist degradation.

Coagulation is caused by the dehydration of proteins through the use of alcohols or acetone. These reagents remove and replace free water in cells and tissues and cause a change in the tertiary structure of proteins by destabilizing hydrophobic bonding. It is also called denaturation.

 

Factors affecting fixation

  1. Temperature: In general, an increase in temperature increased the rate of fixation but also increased the rate of autolysis and diffusion of cellular elements. Traditionally, 0 to 4 °C has been considered the ideal temperature or the fixation of specimens. Now fixation is routinely carried out at room temperature.
  2. Size: 1–4 mm thickness
  3. Volume ratio: At least 15-20 times greater than tissue volume
  4. Time: 24 – 48 hours
  5. pH: Should be kept in the physiological range, between pH 4-9. The pH for the ultrastructure preservation should be buffered between 7.2 to 7.4.

 

Protocols

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