Practical consideration of fixation technique

Experiments have been performed to reveal the effects of different concentrations of aldehyde fixatives on tissue morphology. Over a 10-fold range, varying the concentrations of formaldehyde from 2% to 20% in a fixative solution had little effect on the cytoplasmic volume in one-centimeter blocks of rat kidney biopsies. Only at very high (37-40%) concentrations was there a significant decrease in the cytoplasmic volume. Paraformaldehyde is normally used as 2-3.7% solution, whereas glutaraldehyde is normally used as 1-3% solution. The use of low concentrations of aldehydes, e.g. paraformaldehyde (1%) or glutaraldehyde (0.25%) has been found ideal for immunolabeling purposes, including immuno-fluorescence microscopy and immuno-electron microscopy.

The osmolality of fixatives has a major effect on tissue morphology. Hypertonic solutions cause cell shrinkage. Isotonic fixatives produce swollen cells and poor fixation, as do hypotonic fixatives. The best results were obtained using slightly hypertonic solutions (440-450 mOsm). The vehicle osmolality is more important than the total osmolality of the fixative. Ideally, it should be isotonic with tissues in their normal environment. For aldehyde fixatives, vehicle osmolality should be about 300 mOsm with sodium chloride as vehicle substance.

Fixation is often carried out at room temperature. For immunohistological applications, the temperature range is 0-4^oC. The argument in favor of the lower temperature range are that tissue autolysis is slowed down, as is diffusion of various cellular components. Against this is the fact that chemical reactions involved in fixation are also slower at lower temperatures. In practice, the duration of fixation needs to be extended when lower temperature is used.

Prolonged fixation (>24 hrs) in aldehyde solutions is known to severely inhibit immunological reactions. This can be attributed to extensive crosslink formation between protein molecules. Prolonged fixation in formaldehyde has also been shown to cause tissue shrinkage and hardening.

The depth (d) of fixative penetrated into a tissue is proportional to the square root of time (t), which can be written as: