High-pressure freezing combined with in vivo-DAB-cytochemistry: a novel approach for studies of endocytic compartments.
Methods for fine structural and functional analyses of complex and dynamic cell compartments must ensure high temporal resolution together with an excellent fine structural preservation. High-pressure freezing followed by freeze-substitution, and resin embedding is state of the art but its use is limited in combination with preembedding cytochemical techniques. Here we show a new approach for the exploration of compartments of the endocytosis system, which combines high-pressure freezing with peroxidase-catalyzed cytochemistry, thus using the potencies of both synergistically. Uptake of horseradish peroxidase-labeled molecules is followed by in vivo-staining and immobilization of endocytic compartments by generation of diaminobenzidine precipitates. Subsequently, the specimens are high pressure frozen, freeze-substituted, and embedded in resin. The excellent fine structural preservation, together with the high temporal resolution, and differentiating visualization of endocytic compartments qualify the new approach for morpho-functional studies of the complex and dynamic components of the endocytosis system involved in physiologic and pathologic cellular traffic, and in routes utilized in drug targeting strategies. The distinct appearances of membranes and reactive compartments provide optimal conditions for 3D-analyses by electron tomography allowing to discern subtle details of the complex 3D-structures of endocytic compartments.
Department of Cell Biology and Ultrastructure Research, Center for Anatomy and Cell Biology, Medical University of Vienna, Schwarzspanierstrasse 17, A-1090 Vienna, Austria.
Adolf
A
Ellinger
Monika
M
Vetterlein
Christoph
C
Weiss
Claudia
C
Meisslitzer-Ruppitsch
Josef
J
Neumüller
Margit
M
Pavelka
D055032:Electron Microscope Tomography; D004705:Endocytosis / Q000502:physiology*; D005615:Freezing; D006056:Golgi Apparatus / Q000378:metabolism; D056945:Hep G2 Cells; D006651:Histocytochemistry / Q000379:methods*; D006735:Horseradish Peroxidase / Q000378:metabolism; D006801:Humans; D011312:Pressure
D006735:Horseradish Peroxidase