Improved method for freeze-fracture radioautography of tissues and cells, as applied to duodenal epithelium and thymic lymphocytes
An improved method has been devised for the localization of radioactive substances to either one of the leaflets of cellular membranes. After tissue specimens are freeze-fractured and covered with a platinum-carbon replica, they are freeze-dried to allow coating with radioautographic emulsion at room temperature. After exposure at 4 degrees C and development, the emulsion is protected by layers of carbon and grease before the tissue underlying the replica is dissolved in sodium hypochlorite. The grease is removed in Freon 14 and the replica with its emulsion cover is mounted on a specimen grid for electron microscopic examination. The accuracy of radioactivity localization was demonstrated using /sup 3/H-thymidine-labeled liver by finding silver grains over the same sites after freeze-fracture as after thin section radioautography. Tests with /sup 3/H-methacrylate revealed that the interposition of a platinum-carbon replica decreased the radioautographic reaction by over 80%; hence, the need for long exposure. Only 67% of the silver grains came from radiation sources located beyond the upper 0.05 micron of the specimen and, therefore, the emulsion could be affected by radiation sources located not only within membrane leaflets but also in nearby cytoplasm. Thus, when /sup 3/H-fucose was injected into rats to locate newly formed glycoproteins within intestinal epithelium membranes, some of the silver grains found over E and P faces might be produced by radiation coming from the adjacent cytoplasm. To localize label within membrane leaflets in the absence of radiation sources in the cytoplasm, lymphocyte suspensions were incubated with /sup 3/H-concanavalin A at 0 degrees C. The plasmalemma radioactivity was then restricted to the two membrane leaflets, with 87-93% of the silver grains on the E leaflet and 7-13% on the P leaflet.
- Research Organization:
- McGill Univ., Montreal, Quebec
- OSTI ID:
- 6540161
- Journal Information:
- J. Histochem. Cytochem.; (United States), Vol. 32:1
- Country of Publication:
- United States
- Language:
- English
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CELL MEMBRANES
AUTORADIOGRAPHY
CYTOLOGY
ELECTRON MICROSCOPY
TRITIUM COMPOUNDS
BIOLOGICAL LOCALIZATION
ANIMAL CELLS
EPITHELIUM
LYMPHOCYTES
LYOPHILIZATION
SAMPLE PREPARATION
SMALL INTESTINE
ANIMAL TISSUES
BIOLOGICAL MATERIALS
BIOLOGY
BLOOD
BLOOD CELLS
BODY
BODY FLUIDS
CELL CONSTITUENTS
CONNECTIVE TISSUE CELLS
DIGESTIVE SYSTEM
GASTROINTESTINAL TRACT
INTESTINES
LABELLED COMPOUNDS
LEUKOCYTES
MATERIALS
MEMBRANES
MICROSCOPY
ORGANS
SOMATIC CELLS
TISSUES
551001* - Physiological Systems- Tracer Techniques