Fixation for EM – Optical Analysis Facility

Specimen Preparation

Fixation time and temperature depend on the nature and size of the specimen and the composition of the fixative.
Fixation time should be long enough for the stabilizing action of the fixative to be complete, but not long enough to cause excessive extraction of components. The time depends on the rate of penetration of the fixative and the size of the specimen.
Fixation temperature depends on the desired results and the nature of the fixative.
- In general, the penetration rate of the fixative is higher at room or body temperature.
- Low temperature (0-4° C) slows down autolytic changes and reduces shrinkage of specimens, but some labile cell structures such as cytoplasmic microtubules may be lost.
- Fixation in glutaraldehyde may be performed at room temperature or in the cold.
- Fixation of tissue blocks in osmium tetroxide is usually performed in the cold.
- Fixation of single cells or very small specimens in osmium tetroxide may be performed at room temperature.

Specimens must be washed sufficiently between treatments to eliminate free fixative or stain. Keep the following in mind:

Unreacted polymerized glutaraldehyde may cause a pepper-like precipitate (small black dots) when exposed to osmium tetroxide.
Residual osmium tetroxide will react with unpreserved lipids extracted during dehydration.
Uranyl acetate will precipitate in the presence of phosphate or cacodylate buffer.
The solubility of uranyl acetate is reduced at higher concentrations of ethanol or acetone.

If fixation and washing have been carried out at 0-4° C, dehydration should be carried out in the cold.

Infiltration is always performed at room temperature due to the increased viscosity of resins at colder temperatures.
The duration of each step should be sufficient to allow complete infiltration of the embedding medium and depends on the density and size of the specimen as well as the viscosity of the resin.