Sample size for high pressure freezing is limited as vitrification is effective only to the depth of 200 µm. Therefore a very small and thin piece of tissue or a very small volume (several microlitres) of cell suspension is required. A solution of cryoprotectant, most commonly an animal serum albumin, is used to surround the sample during the freezing to prevent drying out and ice crystal formation and optimise heat transfer.
Following the freezing, vitrified water is removed with solvent containing one or a combination of the following components to increase contrast and additionally fix the specimen: a heavy metal stain (most commonly uranyl acetate), formaldehyde, glutaraldehyde, osmium tetroxide, tannic acid. This process is known as freeze substitution. The solvent is finally replaced with liquid resin and cured at sub-zero temperature using UV light to avoid heat damage to epitopes.