High-pressure freezing and freeze substitution of gravid Caenorhabditis elegans (Nematoda: Rhabditida) for transmission electron microscopy

Abstract

Abstract Because chemical fixatives have a profound negative influence on tissue morphology and antigenicity, alternative fixation methods must be applied for some purposes. In this work we used high-pressure freezing (HPF) followed by freeze substitution to maximally preserve antigenicity and morphology. We developed a pipette method for bringing living Caenorhabditis elegans nematodes into the HPF recipient. Using cellulose tubes, it is possible to select individual nematodes for fixation. We were able to HPF complete adults and preserve the morphology in an enhanced fashion compared to chemically fixed tissue. Cellular organelles, especially mitochondria, were much better preserved. Uterine embryos protected by the intact eggshell were excellently preserved without the need for elaborate techniques. Antigenicity with MH27 and ICB4 antisera was tested. With the MH27 serum, an adequate, reproducible, specific binding pattern with chemically fixed tissue could only be achieved using purified antibodies, whereas with highpressure freezing, unpurified MH27 antisera was effective. For ICB4 antisera, a reproducible specific binding pattern was achieved at a concentration of primary antiserum 1000 × lower than that for chemically fixed tissue.