Fig. 3.

Staining of Spc42-2xMTH with a gold enhancement kit. Yeast cells expressing the tagged version of SPC42 were high-pressure frozen, freeze-substitution fixed in acetone with low concentrations of glutaraldehyde or uranyl acetate and embedded in K4M. (A) Sections incubated with aurothiomalate alone did not show appreciable staining (Table S1, condition #8). (B) Labelling was observed on sections stained only with the Nanoprobes gold enhancement solutions for 5 min at room temperature (Table S1, condition #7). Note that the heavy metal in this protocol has worked as a stain for ribosomes and spindle microtubules as well as the band of Spc42, emphasizing the problem of SNR in obtaining convincing localizations in the EM. (C) A better SNR was achieved by staining first with aurothiomalate for 1 h at 37°C, then enhancing that gold deposition with the Nanoprobes kit (Table S1, condition #3). Bars = 100 nm.