Nematology 19 (2017) 871-881 brill.com/nemy Self-Pressurised Rapid Freezing (SPRF): an easy-to-use and low-cost alternative cryo-fixation method for nematodes Myriam CLAEYS 1,∗ , Vladimir V. YUSHIN 2,3 , Jan L.M. L EUNISSEN 4 , Jef CLAEYS 1 and Wim BERT 1 1 Nematology Research Unit, Department of Biology, Ghent University, Belgium 2 National Scientific Center of Marine Biology, Far Eastern Branch, Russian Academy of Sciences, Vladivostok 690041, Russia 3 Far Eastern Federal University, Vladivostok 690950, Russia 4 Aurion Immuno Gold Reagents & Accessories, Wageningen, The Netherlands Received: 31 May 2017; revised: 14 July 2017 Accepted for publication: 15 July 2017; available online: 17 August 2017 Summary – Self-Pressurised Rapid Freezing (SPRF), an easy-to-use and low-cost alternative cryo-fixation method, was evaluated based on a comparative analysis of the ultrastructure of spermatozoa of the nematodes Acrobeles complexus and Caenorhabditis elegans. Sealed copper tubes, packed with active nematodes in water, were plunged into nitrogen slush, a semi-solid form of nitrogen. The water inside the capillary copper tube expands upon cooling due to the formation of hexagonal ice, thereby generating high pressure intrinsically for cryo-fixation of the sample. For sperm cells cryo-fixed by SPRF, the preservation of the ultrastructure was comparable to that achieved with high pressure freezing. This was evidenced by the clear details in mitochondria, membranous organelles and cytoskeleton in the pseudopod. It was demonstrated that SPRF fixation did not destroy antigenicity, based on the results of the immunolocalisation of the major sperm protein in both species. In conclusion, SPRF is a low-cost alternative cryo-fixation method for nematodes. Keywords – Acrobeles complexus, Caenorhabditis elegans, freeze substitution, high-pressure freezing, immunogold labelling, major sperm protein, MSP, spermatozoa, TEM. For over 50 years transmission electron microscopy (TEM) has been a crucial method for studying tissues and cells of nematodes. The ultrastructural information on cuticular derivatives, nervous system and sensory or- gans, somatic tissues and cell types, gonads and gametes, as well as morphogenesis in embryos, germ cell develop- ment and moulting, have been comprehensively described through the use of TEM (Bird & Bird, 1991; Wright, 1991; Malakhov, 1994; Hall & Altun, 2008). In these studies chemical fixation was the standard method for tissue preservation. However, the rapid fixation offered by cryomethods is indispensable for high resolution and advanced approaches, including immunocytochemistry, tomography and 3D reconstruction, quantitative assess- ments of cytoplasmic elements, and correlative light and electron microscopy (CLEM) (Bumbarger et al., 2006; Hurbain & Sachse, 2011; McDonald, 2014a; Mielanczyk et al., 2014; Bert et al., 2016). Cryo-fixation is faster ∗ Corresponding author, e-mail: nini.claeys@ugent.be than most of the cellular processes, and cryo-preparation in toto provides structural preservation closer to the na- tive state, causing fewer artifacts to occur in cell and or- ganelle morphology. Cryo-preparation methods are cur- rently also the preferred way to preserve tissue antigenic- ity for immuno-electron microscopy (Claeys et al., 2004; Rostaing et al., 2004; Weimer, 2006; Morphew, 2007; Hall et al., 2012; Yushin et al., 2016). High pressure freezing and freeze substitution is a cryo- fixation method often used in cell and developmental bi- ology, and it is also widely applied to fixation of the ne- matode Caenorhabditis elegans as it produces better ul- trastructural quality than conventional chemical fixation (Rostaing et al., 2004; Weimer, 2006; McDonald, 2007, 2014b; Hall et al., 2012; Manning & Richmond, 2015; Serwas & Dammermann, 2015). High pressure freezing (HPF) is based on the solidification of water in biolog- ical objects. By cooling down the specimens rapidly in © Koninklijke Brill NV, Leiden, 2017 DOI 10.1163/15685411-00003093