Freeze-Sectioning of Plant Tissues

Abstract

Techniques are described for freeze-sectioning a wide range of both fresh and fixed plant tissues. Gelatin-antifreeze media are used to support but not infiltrate the tissue during sectioning. At cryostat temperatures of -10 to -15 C, 15% gelatin (w/v) containing 0.8% dimethyl sulfoxide (DMSO), or 1.5% ethanediol (ethylene glycol), or 2% glycerol is used. Lower concentrations of gelatin and higher concentrations of antifreezes are required for sectioning at -24 C. Petri plates of media are stored at 2 C, and used by simply melting a hole in the medium. Fresh tissues can be placed directly in the hole, or prefrozen at temperature of liquid nitrogen, or equilibrated in antifreeze solution, before freeze-sectioning in the gelatin antifreeze medium. Many plant tissues have highly vacuolated cells and need equilibration in antifreeze solutions prior to freeze-sectioning. Fixed tissues are rehydrated and washed in water or buffer for 15-24 hr before equilibrating in a 10% solution of either DMSO, ethanediol or glycerol (named in order of rapidity of equilibration). Pretreatment in 10% DMSO is usually for 1-6 hr at 2 C for histochemical studies; or in 10% ethanediol or glycerol for 15-24 hr at either room temperature or 37 C for morphological studies. These methods permit serial cryostat sections free from freezing and thawing artifacts to be cut as thin as 2 μ.