Low-temperature embedding

The maintenance of low temperatures during the processing of fixed specimens is a valuable aid to the retention of soluble components close to their in vivo positions. The production of a resin that can be infiltrated at low-temperatures and also polymerised, also at sub-zero temperatures, was clearly a major advance. The Lowicryl resins were developed in the 1980's for this purpose. Now there are also the London Resin Company resins: LR White and LR Gold, but these can only be used down to about -20⁰C.

There are essentially two different processing routes for embedding in these acrylic resins: firstly, via normal (ambient temperature) fixation with subsequent, gradual, lowering of temperature during the dehydration stages; secondly, using freeze-substitution so that the cryo-fixed, frozen specimen is never warmed until it is fully infiltrated with resin and polymerized. The first pathway is the most frequently used and it is the method of choice at the John Innes Centre; fixing in glutaraldehyde at room temperature, dehydrating in ethanol down to -35⁰C, followed by infiltration with, and polymerisation of, LR White at -20⁰C. Since this route involves chemical fixation rather than cryo-fixation, it may not strictly be regarded as a cryo-technique, but the fact that it involves sub-zero temperatures for part of it's protocol, in order to preserve antigenicity, means that it is generally included under the banner of cryo-methodology.