In situ localization of cholesterol in skeletal muscle by use of a monoclonal antibody

A common perception is that cholesterol, the major structural lipid found i n mammalian membranes, is localized nearly exclusively to the plasma membra ne of living cells and that it is found in much smaller quantities in inter nal membranes. This perception is based almost exclusively on cell fraction ation studies, in which density gradient centrifugation is used for purific ation of discrete subcellular membrane fractions. Here we describe a monocl onal antibody, MAb 2C5-6, previously reported to detect purified cholestero l in synthetic membranes (Swartz GM Jr, Gentry MK, Amende LM, Blanchette-Ma ckie EJ, and Alving CR. Proc Natl Acad Sci USA 85: 1902-1906, 1988), that i s capable of detecting cholesterol in situ in the membranes of skeletal mus cle sections. Localization of cholesterol, the dihydropyridine receptor of the T tubule, and the Ca2+-ATPase of the sarcoplasmic reticulum (SERCA2) by means of double and triple immunostaining protocols clearly demonstrates t hat cholesterol is primarily localized to the sarcoplasmic reticulum membra nes of skeletal muscle rather than the sarcolemmal or T tubule membranes. T he availability of this reagent and its ability to spatially localize chole sterol in situ may provide a greater understanding of the relationship betw een membrane cholesterol content and transmembrane signaling in skeletal mu scle.