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The immunogold method is widely used to localize, identify, and distinguish cellular antigens. There are, however, some pitfalls that can lead to nonspecific binding, particularly in cytoskeletal studies with gold probes prepared from small gold partides. We present a list of suggestions for minimizing nonspecific binding, with particular attention to two problems identified in this study. First, we find that the method used to prepare the colloidal gold partides aSfects the degree of nonspecific binding. Second, the standard BSA-stabilized small gold probes evidently possess exposed regions that bind to the proteins of cytoskeletal preparations. This w s investigated in whole-mount cytoskeletal preparations ofcultured cells by use oflight microscopy, transmission electron microscopy, and photoelectron microscopy ofsilver-enhanced specimens. Gold probes were made BIRRELL, HEDBERG, GRIFFITh screening of the various preparations by light microscopy (LM) before they are examined by electron microscopy (EM). We also make use ofthe relatively new, highly surface-sensitive technique of photoelectron microscopy (Griffith and Rempfer, 1985), in addition to conventional TEM, in our analysis ofthe effectiveness of various procedures to increase the specificity of colloidal gold labeling. Materials and Methods Preparation ofColloidal Gold. Small colloidal gold particles prepared by several different procedures were used in this study: (a) 5-nm colloidal gold made with white phosphorus was obtained fromJanssen Pharmaceutica (Beerse, Belgium); (b) 4-5-nm colloidal gold, prepared using sodium borohydride as the reducing agent, was made by the following procedure (Tschopp et al., 1982): to 40 ml of distilled water at CC containing 0.5 ml of 1% HAuCl (Sigma; St. Louis, MO) and 200 tl of0.2 M K2C03 was added 400-gil aliquots ofNaBH. until the color changed from blue-purple to red-orange; (c) 4-6-nm colloidal gold was made according to the citrate-tannic acid procedure (Slot and Geuze, 1985): to a 125-mI flask was added 79 ml distilled water plus 1 ml 1% HAuCl.. To a 50-mi beaker were added 6 ml distilled water, 4 ml 1% trisodium citrate, 5 ml 1% tannic acid (low molecular weight galloylglucose; Mallinckrodt, Paris, KY) and 5 ml 25 mM K2CO3. Both vesselswere heated to 60C, and the mixture of reducing agents was added, with very rapid stirring, to the gold chloride solution. The mixture was then heated to boiling. Colloidal gold preparations were brought to pH 9.0 with 0.2 M K2CO, before complex formation with antibodies. Preparation ofAntibody-Gold Complexes. Affinity-purified goat antimouse 1gM was obtained from HyClone Laboratories (Logan, UT) in PBS and was first diluted to 0.3 mg/mI with 3 M KCNS, dialyzed against 3

Several studies have indicated that olfactory responses are impeded by amiloride. Therefore, it was of interest to see whether, and if so which, olfactory epithelial cellular compartments have amiloride-sensitive structures. Using ultrastructural methods that involved rapid freezing, freeze-substitution and low temperature embedding of olfactory epithelia, this study shows that, in the rat, this tissue is immunoreactive to antibodies against amiloride sensitive Na(+)-channels. However, microvilli of olfactory supporting cells, as opposed to receptor cilia, contained most of the immunoreactive sites. Apices from which the microvilli sprout and receptor cell dendritic knobs had much less if any of the amiloride-antibody binding sites. Using a direct ligand-binding cytochemical method, this study also confirms earlier ones that showed that olfactory receptor cell cilia have Na+, K(+)-ATPase. It is proposed that supporting cell microvilli and the receptor cilia themselves have mechanisms, different but likely complementary, that participate in regulating the salt concentration around the receptor cell cilia. In this way, both structures help to provide the ambient mucous environment for receptor cells to function properly. This regulation of the salt concentration of an ambient fluid environment is a function that the olfactory epithelium shares with cells of transporting epithelia, such as those of kidney.

The extrinsic membrane protein cytochrome c binds to lipid mixtures containing negatively charged phospholipids such as diphosphatidylglycerol (DPG). In this study the effect of cytochrome c on the lipid distribution in a DPG-steroid spin-label (3-doxyl-5alpha-cholestane) model membrane system is examined. The electron spin resonance (ESR) line-shape changes indicate that cytochrome c induces lateral phase separation at room temperature. The resulting two-dimensional lipid distribution is nonrandom, consisting of clusters of phospholipids bound to cytochrome c and patches of steroid spin-label molecules. Phase separations are also observed in the three-component system: DPG, phosphatidylcholine, and 3-doxyl-5alpha-cholestane.