The objective of the present study was to develop a stain permitting automated quantification of myocellular lipid depositions in skeletal muscle sections together with immunolocalisation of other myocellular constituents by fluorescence microscopy. Lipid droplets were detected in skeletal muscle by oil red O (ORO). Conventional ORO was modified to diminish background staining, prevent crystallisation of ORO and to optimise lipid retention in cryosections. These modifications resulted in a punctate staining of lipid droplets, rather than the somewhat diffuse staining by conventional ORO. Small cavities in muscle sections (like the lumen of small blood vessels) lack ORO when using the protocol presented here. In addition a staining protocol is presented combining ORO with immunofluorescence. This combination permits multiple staining studies in the same section. Thus, lipid droplets can be studied together with immunolabelling of proteins involved in lipid handling and metabolism. This will extend our knowledge on the subcellular localisation of lipid handling proteins (i.e. enzymes and fatty acid transporting proteins) in relation to the localisation of lipid depositions. In conclusion, the protocol presented here permits examination of ORO-stained lipid droplets in skeletal muscle sections together with multiple staining of other immunodetectable proteins present in skeletal muscle by quantitative fluorescence microscopy.