[Electron microscopic demonstration of pulmonary surfactant proteins using procion brilliant blue H5GS]

A new electron microscopy technique is described for detection of lung surfactant proteins with the copper-containing phthalocyanine dye, procion brilliant blue H5GS. The protein structures were stained concurrently with the fixation during perfusion through the pulmonary artery of a fixative-staining mixture containing glutaric aldehyde and a dye in the kakodilate buffer, pH 5.6-6, and in the course of a subsequent immersion of lung tissue pieces into the same mixture. Then the material was treated with thiosemicarbazide and post-fixed with OsO4. The dye did not penetrate intact cells. The electron-dense products of the histochemical reaction were located inside and on the surface of the surfactant membrane, in the hypophase of the surfactant complex, on the plasmalemma of air-blood barrier cells and in its micropinocytosis vesicles, as well as on the membranes of osmophilic plate-like bodies as their contents egressed into the alveolar lumen.