Chemical Fixation

To be able to view a biological sample in the electron microscope it must first be stabilized or "fixed", preferrably in a way that the ultrastructure of the cells or tissue remain as close to the living material as possible. The choice of fixative depends on the purpose of your study. While finding the right fixation conditions is often a matter of trial and error, standard protocols can be sufficient for many purposes. Below are some of the most common chemicals used in routine TEM fixation.

Glutaraldehyde reacts with many nucleophiles in the cell (most commonly amines). It produces irreversible cross-linking networks throughout the cytoplasm in seconds to minutes. The reaction results in a drop in pH from a significant release of protons, making adequate buffering important. Note that a too high concentration of glutaraldehyde can inhibit the formation of rapid cross-links.

Formaldehyde cross-links amino groups of proteins. The reaction is much slower than that of glutaraldehyde, normally using a 2-8% solution you need to fix for at least 2 hours at RT. Due to the drop in pH during the reaction of formaldehyde and amino groups adequate buffering is important.

Glutaraldehyde-formaldehyde mixtures. The original paper by Karnovsky (1965) recommends a mixture of 4% glutaraldehyde and 6% formaldehyde. Other empirically determined mixtures are widely used for both structural and immunocytochemical studies. We routinely use a mixture of  2% formaldehyde and 2.5 % glutaraldehyde in 0.1 M Sodium Cacodylate buffer, pH 7.4.

Acrolein is a very toxic and highly volatile reactive aldehyde that's mostly been used in mixtures with glutaraldehyde and/or formaldehyde. Cross-links proteins as rapidly as glutaraldehyde.

Osmium tetroxide is normally used as a secondary fixative after formaldehyde/glutaraldehyde. It reacts with unsaturated acyl chains of membrane lipids and nucleophiles like amino and sulphhydryl groups. While its main purpose is contrasting it also increases the retention of lipids in the tissue.

Tannic Acid is a mixture of polyglycol anions that preserve tissue via non-covalent interactions. Can be used after primary fixation with an aldehydes to enhance contrast and preserve antigenicity. Makes a good substitute for Osmium tetroxide when used in combination with 1% Uranyl Acetate.