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In this chapter an overview of the most commonly used techniques in both quantitative and qualitative antifreeze protein (AFP) research is presented. This includes, among others, thermal hysteresis measurement by nanoliter cryoscopy, ice recrystallization inhibition assay by microscopy and immunolocalization. AFP research has come a long way, from merely observing the effects of AFP to explaining some of the fundamental mechanisms of action. These involve a dynamic hydration shell around proteins, the nanometer curvature of ice crystals in the presence of AFP, and the flux of water molecules between ice crystals as they recrystallize. The interest in AFP as an additive to food has always been high, and a wealth of methods have been applied to foods with added AFP. Most of these are not concerned with the function of AFP, but rather the effect on a complex matrix, such as meat or dough. Some of these indirect methods for detecting the effects of AFP are also presented at the end of the chapter.

The aim of this study is to provide the reader with a simple setup that can detect antifreeze proteins (AFP) by inhibition of ice recrystallisation in very small sample sizes. This includes an open source cryostage, a method for preparing and loading samples as well as a software analysis method. The entire setup was tested using hyperactive AFP from the cerambycid beetle, Rhagium mordax. Samples containing AFP were compared to buffer samples, and the results are visualised as crystal radius evolution over time and in absolute change over 30 min. Statistical analysis showed that samples containing AFP could reliably be told apart from controls after only two minutes of recrystallisation. The goal of providing a fast, cheap and easy method for detecting antifreeze proteins in solution was met, and further development of the system can be followed at https://github.com/pechano/cryostage.

Larvae of the blackspotted pliers support beetle, Rhagium mordax, were collected monthly, for the duration of 2012 and fixed. The larvae were embedded in paraffin wax and sectioned. Using fluorophore-coupled antibodies specific to the R. mordax antifreeze protein, RmAFP1, sections were visualised with UV reflected light microscopy. An automated software analysis method was developed in order to discard autofluorescence, and quantify fluorescence from bound antibodies. The results show that R. mordax cuticle and gut exhibit a higher degree of fluorophore-bound fluorescence during summer, than in the cold months. It is hypothesised that R. mordax stores RmAFP1 in, or near, the fat body during times when freeze avoidance is not needed.