Transmission electron microscopy and immunocytochemical studies of yeast: analysis of HMG-CoA reductase overproduction by electron microscopy.
The results and anecdotes presented here are intended only as a general guide to other would-be immunocytochemists, because other proteins will undoubtedly respond at least somewhat differently than does HMG-CoA reductase. Nevertheless, based on these experiences, we offer the following suggestions: 1. Antiserum of high specificity should be raised and affinity-purified. Using this antiserum, immunofluorescence microscopy should be attempted before resorting to electron microscopic localization. In the absence of immunolocalization at the light-microscope level, it may be a waste of time to pursue the problem to higher levels of resolution. 2. Cells should be prefixed in 1% formaldehyde-1% glutaraldehyde. Direct fixation of the growing culture and use of phosphate buffer are recommended. The prefixed sample can then be divided into two or three aliquots. One aliquot should receive no postfixation (for optimal immunoreactivity), while the others can be postfixed in osmium-potassium ferricyanide (for possible immunolocalization) or permanganate (for ultrastructural analysis). Because of its ease of use, Spurr's resin should be tried initially. If immunocytochemistry is successful, no further preparations are necessary. If unsuccessful, LR White resin is recommended, but the sample must be treated to remove the cell wall. Electron microscopy and immunocytochemistry offer views into the molecular arrangement of individual cells, a view not easily obtained by other means. It is satisfying and often enlightening to be able to see the extremes as well as the average. In studies of the organization of karmellae, for example, ultrastructural analysis easily revealed the asymmetric segregation pattern, while immunoblots and cell fractionation could not even demonstrate the existence of this membrane organization. The richness of the information available to those who can avert reductionist tendencies, even for a short time, is remarkable.
Wright
R
R
Department of Biochemistry, University of California, Berkeley 94720.
Rine
J
J
D006651:Histocytochemistry; D006903:Hydroxymethylglutaryl CoA Reductases / Q000096:biosynthesis*; D007150:Immunohistochemistry / Q000379:methods; D007202:Indicators and Reagents; D008854:Microscopy, Electron / Q000379:methods; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000648:ultrastructure
D007202:Indicators and Reagents; D006903:Hydroxymethylglutaryl CoA Reductases