Labelled-replica techniques: post-shadow labelling of intramembrane particles in freeze-fracture replicas. 1982

J E Rash, and T J Johnson, and C S Hudson, and F D Giddings, and W F Graham, and M E Eldefrawi

Three methods are described for direct post-fracture, post-shadow labelling of individual classes of intramembrane particles (IMPs) in freeze-fracture replicas of biological membranes. The P-face IMPs corresponding to the acetylcholine receptor complexes (AChRs) of vertebrate neuroeffector junctions are identified by post-replication labelling with ferritin-antibody complexes and with neurotoxin-biotin-avidin-colloidal gold affinity ligands. (The freeze-etch nomenclature of Branton et al., 1975, is used in this report.) These post-shadow labelling techniques resemble conventional en bloc labelling techniques except that the labelling reagents must penetrate a thin but discontinuous layer of platinum superimposed on the molecules of interest. In the 'sectioned labelled-replica technique', the replicated and labelled tissues are stained, embedded in plastic and sectioned parallel to the replica-tissue interfaces. In the direct 'labelled-replica techniques', the replicated and labelled samples are freeze-dried or critical point dried, the labelled surfaces are stabilized by carbon coating, and the underlying tissues are dissolved, allowing the labelled-replicas to be examined as conventional freeze-fracture replicas. The unshadowed side of each AChR IMP is shown to retain sufficient biochemical information to permit both immunospecific and neurotoxin specific labelling despite formaldehyde fixation, freezing, fracturing, platinum shadowing, and thawing in aqueous media. A new mixed ferricyanide-osmium staining method reveals electron opaque structures spanning the membrane bilayer in the same size, number and distribution as the labelled IMPs. These experiments demonstrate the feasibility of identifying individual IMPs in freeze-fracture replicas and may allow the identification of specific membrane lesions in human disease.

UI MeSH Term Description Entries
D007425 Intracellular Membranes Thin structures that encapsulate subcellular structures or ORGANELLES in EUKARYOTIC CELLS. They include a variety of membranes associated with the CELL NUCLEUS; the MITOCHONDRIA; the GOLGI APPARATUS; the ENDOPLASMIC RETICULUM; LYSOSOMES; PLASTIDS; and VACUOLES. Membranes, Intracellular,Intracellular Membrane,Membrane, Intracellular
D008565 Membrane Proteins Proteins which are found in membranes including cellular and intracellular membranes. They consist of two types, peripheral and integral proteins. They include most membrane-associated enzymes, antigenic proteins, transport proteins, and drug, hormone, and lectin receptors. Cell Membrane Protein,Cell Membrane Proteins,Cell Surface Protein,Cell Surface Proteins,Integral Membrane Proteins,Membrane-Associated Protein,Surface Protein,Surface Proteins,Integral Membrane Protein,Membrane Protein,Membrane-Associated Proteins,Membrane Associated Protein,Membrane Associated Proteins,Membrane Protein, Cell,Membrane Protein, Integral,Membrane Proteins, Integral,Protein, Cell Membrane,Protein, Cell Surface,Protein, Integral Membrane,Protein, Membrane,Protein, Membrane-Associated,Protein, Surface,Proteins, Cell Membrane,Proteins, Cell Surface,Proteins, Integral Membrane,Proteins, Membrane,Proteins, Membrane-Associated,Proteins, Surface,Surface Protein, Cell
D008854 Microscopy, Electron Microscopy using an electron beam, instead of light, to visualize the sample, thereby allowing much greater magnification. The interactions of ELECTRONS with specimens are used to provide information about the fine structure of that specimen. In TRANSMISSION ELECTRON MICROSCOPY the reactions of the electrons that are transmitted through the specimen are imaged. In SCANNING ELECTRON MICROSCOPY an electron beam falls at a non-normal angle on the specimen and the image is derived from the reactions occurring above the plane of the specimen. Electron Microscopy
D009469 Neuromuscular Junction The synapse between a neuron and a muscle. Myoneural Junction,Nerve-Muscle Preparation,Junction, Myoneural,Junction, Neuromuscular,Junctions, Myoneural,Junctions, Neuromuscular,Myoneural Junctions,Nerve Muscle Preparation,Nerve-Muscle Preparations,Neuromuscular Junctions,Preparation, Nerve-Muscle,Preparations, Nerve-Muscle
D011950 Receptors, Cholinergic Cell surface proteins that bind acetylcholine with high affinity and trigger intracellular changes influencing the behavior of cells. Cholinergic receptors are divided into two major classes, muscarinic and nicotinic, based originally on their affinity for nicotine and muscarine. Each group is further subdivided based on pharmacology, location, mode of action, and/or molecular biology. ACh Receptor,Acetylcholine Receptor,Acetylcholine Receptors,Cholinergic Receptor,Cholinergic Receptors,Cholinoceptive Sites,Cholinoceptor,Cholinoceptors,Receptors, Acetylcholine,ACh Receptors,Receptors, ACh,Receptor, ACh,Receptor, Acetylcholine,Receptor, Cholinergic,Sites, Cholinoceptive
D002244 Carbon A nonmetallic element with atomic symbol C, atomic number 6, and atomic weight [12.0096; 12.0116]. It may occur as several different allotropes including DIAMOND; CHARCOAL; and GRAPHITE; and as SOOT from incompletely burned fuel. Carbon-12,Vitreous Carbon,Carbon 12,Carbon, Vitreous
D005293 Ferritins Iron-containing proteins that are widely distributed in animals, plants, and microorganisms. Their major function is to store IRON in a nontoxic bioavailable form. Each ferritin molecule consists of ferric iron in a hollow protein shell (APOFERRITINS) made of 24 subunits of various sequences depending on the species and tissue types. Basic Isoferritin,Ferritin,Isoferritin,Isoferritin, Basic
D005612 Freeze Drying Method of tissue preparation in which the tissue specimen is frozen and then dehydrated at low temperature in a high vacuum. This method is also used for dehydrating pharmaceutical and food products. Lyophilization,Drying, Freeze,Dryings, Freeze,Freeze Dryings,Lyophilizations
D005614 Freeze Fracturing Preparation for electron microscopy of minute replicas of exposed surfaces of the cell which have been ruptured in the frozen state. The specimen is frozen, then cleaved under high vacuum at the same temperature. The exposed surface is shadowed with carbon and platinum and coated with carbon to obtain a carbon replica. Fracturing, Freeze,Fracturings, Freeze,Freeze Fracturings
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man

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