Easy flat embedding of oriented samples in hydrophilic resin (LR White) under controlled atmosphere: Application allowing both nucleic acid hybridizations (CARD-FISH) and ultrastructural observations

Summary hilic resins present the advantage of making possible both hybridization experiments involving either antibodies or oligonucleotide probes and ultrastructural observations. Whereas various embedding protocols are available, only very few concern flat-embedded preparations. In this study we describe an easy protocol for flat embedding of small-oriented biological samples in hydrophilic resins (LR White). The most important constraints are (i) to polymerize the samples under argon-saturated atmosphere (avoiding oxygen which is an inhibitor of LR White polymerization) and (ii) to use transparent flat embedding molds. Two kinds of samples were analyzed: small pieces of large tissue that need to be accurately oriented for a valuable analysis and very small organisms such as free-living nematodes, which are very hard to investigate with conventional paraffin wax embedding techniques. Semi-thin sections strongly reinforce the quality of the observations from oligonucleotidic in situ hybridization experiments by reducing the background usually encountered in oligonucleotide probe hybridization experiments from sections. Such protocols could also permit a cheap alternative to the use of laser scanning confocal microscopes for oligonucleotidic in situ hybridization as in FISH and CARD-FISH experiments from histological sections. The interest of this embedding protocol is reinforced by the fact that molecular in situ hybridization experiments and ultrastructural observations from thin sections can be carried out from a single-small individual (<1 mm in length) sample.