Fixation protocols are usually straightforward. The tissue is cut to dimensions suited to the rate of penetration of the particular fixative and placed in the fixative solution. The number of factors affecting the fixation process includes buffering, penetration, volume, temperature and concentration.

In fixation pH is critical. This is especially the case with formaldehyde, where acidity favors the formation of formalin-heme pigment deposits in tissue and may lead to protein denaturation and structural deformations. Sources of acidity include hypoxia of tissues and oxidation of formaldehyde stocks to formic acid. To prevent extremes of acidity or basicity, fixatives are generally buffered to a pH between 6-8. Common buffers include phosphate, bicarbonate, cacodylate, and veronal. Commercial formaldehyde fixatives are often buffered with phosphate at a pH of 7.

The diffusibility of each fixative will determine its rate of penetration through tissue. Formalin and alcohol penetrate the fastest, glutaraldehyde the slowest, with mercurials and Mirsky’s Fixative falling between these extremes. Normally, slow rates of penetration are only a problem when dealing with whole organ perfusion, because thin tissue sections are easily permeated and not affected by this variable. Speed of fixation can be dramatically improved by the use of microwave protocols.

The standard ratio of fixative to tissue volume is 10:1. Lower volumes can be used if frequent changes of the fixative are carried out to prevent exhaustion. Agitation will enhance the process by ensuring that fresh fixative solution is constantly washing over the surface of the tissue.

Increasing the temperature will increase the speed of fixation, and hot formaldehyde is often used in automated tissue processors. The temperature used must be selected carefully, as thermal denaturation of tissue proteins will begin to occur at extreme temperatures. Generally 60°C is used with formaldehyde fixatives.

The concentration of the fixative can affect the rate of fixation and the total penetration of fixative into the sample. Too high a concentration will lead to hardening of the tissue and the formation of excessive artifacts. Too low a concentration will allow exhaustion of the fixative before the process is complete.

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