Construction of a device composed of common plumbing supplies for freezing microscopy samples.

Freeze fixation of cells and tissues provides a method of avoiding chemical fixation artifacts (9), and it often improves immunolocalization (1,4,9). Various devices and techniques have been devised for freeze fixation of different microbial, plant and animal materials (2,5,6,9,10). One of the simplest methods is plunge freezing, in which samples for microscopy are quickly plunged into a suitable cryogen and held there until completely frozen. Often, liquid propane cooled by liquid nitrogen is used as the cryogen because it has good freezing properties and is relatively inexpensive. High rates of heat transfer are desirable to minimize or avoid crystalline ice formation that damages cellular structure, so copper is a common choice for parts of freezing devices. Commercial or custom-made, machinetooled devices using expensive highgrade, highly polishable copper parts can be used, but we report excellent freezing results using a simple, inexpensive and quickly assembled device that is composed in part of commonly available copper plumbing supplies. For freezing, tobacco seeds [Nicotiana tabacum (L.) cv Xanthi] were germinated on wet filter paper in a humidified chamber for 3 days until the primary root had just broken through the seed coat. Then the seedlings, including the embryos, endosperms and seed coats, were cryoprotected by immersion in 20 mM 2-[N-morpholino]ethanesulfonic acid (MES), pH 5.5, 2 mM MgCl2, 2 mM CaCl2, 2 mM KCl, 0.2 M sucrose for 0.5–1 h (9), cut into 0.5-mm pieces, loaded onto Formvar (Toki, New York, NY, USA) coated 0.3-cm nickel loops and plungefrozen in liquid propane cooled by liquid nitrogen while held using insulated forceps using the device shown in Figure 1. The liquid propane had been produced by blowing a gentle stream of gaseous propane from a commercial blowtorch cylinder into the liquid nitrogen-cooled copper cup (Figure 1). This cup is very smooth to the touch and was easily polished to a high shine using commonly available metal polish. Seedlings were substituted for 3 days at -85°C in either acetone (for electron microscopy) or ethanol (for light microscopy). The samples were then placed in different temperature compartments to gradually warm the samples to room temperature in a stepwise manner: to -20°C overnight, to 4°C over 4 h and to 25°C over 1 h, followed by washes in fresh solvent.