METHODS FOR THE STUDY OF SMALL PHAGOSOMES AND THEIR RELATIONSHIP TO LYSOSOMES WITH HORSERADISH PEROXIDASE AS A "MARKER PROTEIN"

Abstract

Small phagosomes (micropinocytic vesicles and vacuoles) which had taken up injected horseradish peroxidase were identified by staining for peroxidase with benzidine and H₂O₂. Because of the small size of the granules and the possibility of artifacts, previously described procedures had to be modified in several respects. Prefixation of the tissue by perfusion at 37°C prevented artifacts of diffusion and adsorption of peroxidase. The blue product of the reaction of peroxidase with benzidine in the small phagosomes was preserved and fading to brown was prevented by cooling the tissue section to –10° to –15°C during its processing through polar media. The blue reaction product was stable as soon as the section was transferred to an apolar medium. Small phagosomes were visualized together with lysosomes and phago-lysosomes in the same cells by double staining for acid phosphatase and peroxidase in contrasting colors. The incubation for acid phosphatase was performed at 4°C since low temperature increased the stability of peroxidase in the acid medium. Factors which form the basis for other improvements of the procedure are discussed.