HISTOLOGICAL STAINING OF LIPIDS FOR THE LIGHT AND ELECTRON MICROSCOPE

1. It is generally agreed that the blackening of osmium tetroxide by unsaturated lipid is too unpredictable to demonstrate lipid in tissues.
2. At neutral pH osmium tetroxide combines with the double bonds in the lipoproteins of cellular membranes (mitochondria, etc.) and the deep colour reaction of ethyl gallate with this osmium provides good staining of lipid for the light microscope.
3. Osmium taken up by tissue proteins at neutral pH is only a small fraction of that taken up by the lipid. (After acid fixatives osmium tetroxide is a general protein stain.)
4. The uptake of Sudan black B by partition from dilute solution is a specific test for lipid, but in normally fixed tissue most of the structural lipid is 'bound' and is not accessible to the dye.
5. Cautious treatment of fixed tissue with dilute sodium hypochlorite will unmask this lipid for viewing by the light microscope.
6. Direct fixation with neutral osmium tetroxide is an effective method for visualizing lipid for the electron microscope (as in the ethyl gallate method for the light microscope). But the poor penetration of osmium limits its use in this way.
7. After formol/glutaraldehyde fixation much of the lipid in the tissues is 'bound' and does not take up osmium. It can be unmasked by a saturated aqueous solution of thymol.
8. The unmasked lipid can then be rendered more osmiophil by partition in a solution of the highly unsaturated terpene farnesol, thus increasing the uptake of osmium in a renewed application.
9. Some of the novel observations on tissue lipids made by these methods are reviewed.