Multiple labeling in electron microscopy: Its application in cardiovascular research

The heart is a muscular pump kept together by a network of extracellular ma trix components. An increase in collagens, as in chronic congestive heart f ailure (CHF), is thought to have a negative effect on cardiac compliance an d, thus, on the clinical condition. Conventional electron microscopy allows for the study of cellular and extracellular components and scanning electr on microscopy (SEM) can put these structures in three-dimensional perspecti ve. However, in order to study extracellular matrix components in relation to cells, immunoelectron microscopy is superior. We have used this techniqu e in our studies on heart failure. Heart specimens were fixed in 4% parafor maldehyde and 0.1% glutaraldehyde in sodium cacodylate buffer, dehydrated b y the method of progressive lowering of temperature and embedded in LR Gold plastic. Immunolabeling could be achieved with different sized gold-conjug ated secondary antibodies or protein-A gold conjugates. Depending on the ob jective, ultra small gold (USG) conjugates or a regular probe size can be u sed. Labeling efficiency could be increased by bridging antibodies. The dou ble and triple staining procedures were based on single staining methods us ing one and two-face labeling. The choice of antibodies and gold conjugates depended on the objectives. Immunoelectron microscopy, using multiple labe ling, allowed a detailed study of the organization of the extracellular mat rix and its relationship with cardiac myocytes. This may prove to be a usef ul tool for the study of chronic heart failure.