A novel technique for flat-embedding cryofixed plant specimens in LR white resin

Acrylic LR White is used preferentially for many research applications because it possesses unique advantages over other commercially available resin types. LR White has low toxicity; its low viscosity and hydrophilic properties enable it to infiltrate specimens with comparative ease, and specimens embedded in LR White take up stains for light microscopy well and tend to retain superior antigenicity relative to other resins. These qualities make LR White a good choice for immunohistochemistry, light microscopy, and transmission electron microscopy. However, LR White does not heat-polymerize in the presence of oxygen, which precludes its use with many commercially available embedding molds. Furthermore, flat-embedding specimens in LR White is often an arduous task. Therefore, in this paper, we report on the development of novel flat-embedding chambers for use with LR White. Another goal of our studies was the rapid fixation of larger specimens. Chemical fixation was inadequate because of the time required for tissue infiltration, but cryofixation quickly and effectively immobilized intracellular organelles. Mean amyloplast positions differed in vertically oriented versus control specimens after chemical fixation, whereas no significant difference was observed after cryofixation. Furthermore, cryofixation provided acceptable preservation at the light microscopy level, even though our specimens were relatively larger. This underscores the utility of cryofixation for light microscopy determination of organelle positioning within plant cells.