Chemistry: analytical and immunological testing – Including sample preparation – Stabilizing or preserving
Patent
1994-06-01
1997-11-11
Ludlow, Jan
Chemistry: analytical and immunological testing
Including sample preparation
Stabilizing or preserving
118423, 118500, 422100, 422101, 422102, 422104, 427 211, 436174, 436178, G01N 136
Patent
active
056863136
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to a method for the incubation of specimens in fluids, especially for subsequent embedding in capsules by polymerization. The embedding by polymerization, which is preferably carried out at temperatures between -120.degree. C. and +80.degree. C., serves for preparing the specimens for a subsequent preparation of sections for microscopic, especially electron-microscopic, end histochemical investigations.
2. Description of the Related Art
As an alternative to the now current standard methods (chemical fixation in buffered solutions of aldehyde and/or osmiumtetroxide--dehydration in polar organic media--incubation in monomer--embedding in synthetic resin by polymerization; cf. H. Sitte, mta-Extra No. 10, Umschau-Verlag Breidenstein GmbH, Frankfurt-Main, 1985), biological specimens are to an increasing extent frozen in extremely rapidly ("cryofixation"). Subsequently, the ice contained in the specimens is dissolved out at temperatures between about -80.degree. C. and -120.degree. C. by incubation of the frozen specimens in polar organic fluids (for example methanol or acetone) and replaced by these media ("Cryosubstitution: in this connection, cf. inter alia Patent Specifications DE 2,944,464 C2 or DE 3,425,744 C2, or H. Sitte, Zeiss, MEM 3, 25-31, 1984, or H. Sitte et al., GIT Labor-Medizin 10, 199-208, 1987, or H. Sitte et al. in A. J. Verkleij and J. L. M. Leunissen as editors of "Immuno-Gold Labeling in Cell Biology, pages 64-93, and in particular chapter III "Rapid Freezing, Freeze-Substitution and Resin Embedding", CRC-Press, Boca Raton, Fla., USA, 1989; more extensive literature references are included in the latter) and, finally, an embedding by polymerization, initiated by UV irradition, is carried out at low temperature after incubation in a monomer. The alternative "PLT method" (PLT represents "Progressive Lowering of Temperature; in this connection, cf. also embedding at low temperature, inter alia, E. Carlemalm et al., J. Microscopy, Oxford 126, 123-143, 1982, and B. Hurnbel and M. Muller in M. Muller et al., editors of "The Science of Biological Specimen Preparation", SEM Inc., Chicago, pages 175-183, 1986; further literature references are included in the latter) starts from a weak chemical fixation of the specimens (for example in buffered aldehyde) and lowers the temperature of the specimens continuously at steadily increasing concentration of the added polar media (for example methanol) to that extent which corresponds to the freezing point of the particular mixture. This method also concludes with a low-temperature embedding by UV. According to the state of the art, this embedding is as a rule carried out by stepwise transfer of the largely dehydrated specimens into a monomer batch, followed by polymerization by means of UV irradition at temperatures between -30.degree. C. and -70.degree. C.
Both the preparation steps according to the standard method and the said methods at reduced temperature involve a multiple change of the dehydration or substitution media and a stepwise transfer into the pure monomer batch, which has to be carried out individually for each preparation in the case of using Eppendorf tubes for work at reduced temperatures and is extremely time-intensive in the case of relatively large quantities of specimen. A particularly adverse factor is that all individual containers must be sealed gas-tight individually before the UV polymerization at reduced temperature, since otherwise individual components vaporize and perfect curing of the synthetic resin is no longer possible. Further problems are caused by the fact that numerous fixation solutions and substitution media contain powerful poisons having a high vapor pressure (for example the volatile OsO.sub.4) and the usual monomer batches contain powerful allergens, so that both inhalation of the vapors and skin contact can trigger serious illnesses.
SUMMARY OF THE INVENTION
It is the object of the present invention to simplify the embedding in capsu
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Edelmann Ludwig
Sitte Hellmuth
Leica AG
Ludlow Jan
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