A technique for the freeze-fracture of tissue culture. 1978

L Prescott, and M W Brightman

Cultures of rat cerebellum grown on glass coverslips coated first with collodion and then collagen are fixed in glutaraldehyde to which ruthenium red has been added. After fixation, the collodion comes off the glass coverslips and the 3 layers: collodion-collagen-culture are handled as one piece. Following immersion in glycerol, selected areas are cut from the piece; the excised areas are a little smaller than those of the specimen carriers used in a Balzers apparatus. The ruthenium red, which stains all cells and processes, is a good indicator of specimen thickness. The explant which consists of several cell layers is redder than the peripheral monolayer of outgrowth. The square areas cut from either the explant or the outgrowth can be matched in thickness according to their shade of red. In frozen specimens, the red colour distinguishes the culture layer from the collodion-collagen support under it and from the white frost on top. During fracturing, the appearance of pink flakes on the edge of the knife indicates that the culture layer has been cleaved. The growth of cells on collodion-collagen and the fracture of the cultures with a knife, eliminate the restrictions inherent in the use of frozen 'metallic sandwiches'.

UI MeSH Term Description Entries
D002531 Cerebellum The part of brain that lies behind the BRAIN STEM in the posterior base of skull (CRANIAL FOSSA, POSTERIOR). It is also known as the "little brain" with convolutions similar to those of CEREBRAL CORTEX, inner white matter, and deep cerebellar nuclei. Its function is to coordinate voluntary movements, maintain balance, and learn motor skills. Cerebella,Corpus Cerebelli,Parencephalon,Cerebellums,Parencephalons
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
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D046508 Culture Techniques Methods of maintaining or growing biological materials in controlled laboratory conditions. These include the cultures of CELLS; TISSUES; organs; or embryo in vitro. Both animal and plant tissues may be cultured by a variety of methods. Cultures may derive from normal or abnormal tissues, and consist of a single cell type or mixed cell types. Culture Technique,Technique, Culture,Techniques, Culture
D051381 Rats The common name for the genus Rattus. Rattus,Rats, Laboratory,Rats, Norway,Rattus norvegicus,Laboratory Rat,Laboratory Rats,Norway Rat,Norway Rats,Rat,Rat, Laboratory,Rat, Norway,norvegicus, Rattus

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