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1952; BERNHARD W and NANCY MT, 1964; DOLLHOPF FL and SITTE H, 1969; HODSON S and<br />
MARSHALL J, 1970; KOLEHMAINEN-SEVÉUS L, 1970; TOKUYASU KT, 1973; TOKUYASU KT<br />
and SINGER SJ, 1976; TOKUYASU KT, 1986) to the point where this <strong>technique</strong> has become an<br />
appropriate method for ultrastructural studies, it has taken about 20 years of development.<br />
Examples of cryo-<strong>technique</strong>s for immunocytochemical studies include<br />
• Cryo-ultramicrotomy: ultrathin frozen sections (50-70 nm thick) are cut on an<br />
ultramicrotome in a cryochamber. Sections are thawed on grids for transmission<br />
electron microscopy and subsequently immuno-stained, for example with ferritin or<br />
gold conjugated antibodies.<br />
• Cryo-substitution: this is a cryo-variant of conventional resin embedment. The method<br />
avoids denaturing effects of room temperature dehydration and resin embedment.<br />
Samples are frozen, then dehydration and resin embedment are performed in a cryosubstitution<br />
unit, for example at -90°c in acetone. At this temperature the water ice is<br />
replaced by acetone in a sublimation type process which may minimize adverse effects<br />
on antigenicity. The sample is then infiltrated with low viscosity resins (such as the<br />
Lowicryl series) and cured at low temperatures with UV light. Ultrathin sections are<br />
cut from the embedded tissue blocks with conventional ultramicrotomes for<br />
subsequent<br />
immuno-staining.<br />
Apart from the above cryo-<strong>technique</strong>s we have to mention cryo-electron microscopy which is<br />
used to observe molecules, viruses and tissues in their native state without fixation or<br />
staining. A typical application is called frozen hydrated transmission electron microscopy. A<br />
thin layer of unfixed aqueous sample is frozen to give a sheet of vitreous ice with the sample<br />
suspended in it. The preparation is observed at low temperature in the electron microscope<br />
equipped with a special cold stage. Samples prepared in this way are very sensitive to the<br />
electron beam. All immuno-stainings may be done prior to freezing. In a similar way, samples<br />
are prepared for cryo-SEM (scanning electron microscopy) where specimens are rapidly<br />
frozen, sputter coated and transferred to the SEM for imaging.<br />
Selected publications for further readings<br />
Fernández-Morán H (1952)<br />
Bernhard W and Nancy MT (1964)<br />
Dollhopf FL and Sitte H (1969)<br />
Hodson S and Marshall J (1970)<br />
Kolehmainen-Sevéus L (1970)<br />
Kuhlmann WD and Viron A (1972)<br />
Tokuyasu KT (1973)<br />
Tokuyasu KT and Singer SJ (1976)<br />
Tokuyasu KT (1986)<br />
Full version of citations in chapter References.<br />
© Prof. Dr. Wolf D. Kuhlmann, Heidelberg 20.09.2008