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A simple technique for staining of cell membranes with imidazole and osmium tetroxide.

We describe a simple new technique based on the affinity of imidazole and osmium tetroxide for unsaturated lipids. Organs (e.g., kidney, liver, intestine) were perfused in vivo with a glutaraldehyde solution. Tissue fragments were then immersed in a solution containing imidazole and OsO4 and are further stained with a double lead and copper citrate solution. Ultra-thin (0.06 microns) or thick (0.1-0.3 microns) sections were observed with transmission electron microscopy (80-100 kV). The method presented permits excellent visualization of cell membranes (e.g., endoplasmic reticulum, endocytotic apparatus) because it favors good resin penetration and the alkaline pH preserves cell volume. A better stereomicroscopic analysis of the relationship between cell organelles can be carried out with thick sections. The imidazole/osmium can be used routinely because the technical steps are easy and simple to follow. Furthermore, it can complement other cytochemical methods.