Figure 4

Immuno TEM of rat lung labeled for caveolin-1. Caveolae are cholesterol-rich regions of the plasma membrane involved in endocytosis. One of the constituting proteins of caveolae is caveolin-1 which was labeled here using newborn rat lung tissue fixed by instillation of 4% PFA, 0.1% GA in 0.2 M Hepes buffer. After freeze-substitution and embedding in acrylic resin (Table 1), ultrathin sections (40–70 nm) were cut and mounted on formvar-coated Ni mesh grids. Immunogold labeling was performed according to standard protocols [99]. The primary antibody was a rabbit anti-caveolin-1 antibody (BD Biosciences, Pharmingen, Germany) diluted 1:50. The secondary antibody was a goat-anti-rabbit antibody coupled to 10 nm gold particles (British Biocell, Cardiff, United Kingdom). A strong signal is found for caveolae in capillary endothelium and alveolar epithelium. Unspecific background labeling was weak (note the gold particle in the interstitium) but not completely absent. Immunogold labeling requires good knowledge about the biology of the target antigen and the specificity of the antibody. Before going to the TEM level, one is well advised to perform pilot light microscopic experiments. CL = Capillary lumen; EC = Endothelial cell; IC = Interstitial cell; AEI = Alveolar epithelial type I cell; AL = Alveolar lumen. Bar = 1 μm.