A new technique for removal of amorphous phase tissue water without ice crystal damage: a preparative method for ultrastructural analysis and immunoelectron microscopy.

Abstract

An apparatus has been produced that can remove amorphous phase tissue water via molecular distillation without devitrification or rehydration. This method represents a fundamental advance in tissue preparation, making possible for the first time ultrastructural localization of soluble molecular entities without the problems of alteration, re-distribution, and loss which have plagued conventional techniques. Fresh slices of rat brain, liver, or kidney, and monkey retinal tissue were cryofixed by bounce-free, metal mirror cooling on copper bars immersed in liquid nitrogen (LN2). Tissue transferred under LN2 was then placed in a precooled copper specimen block, which was subsequently lowered into a LN2-cooled stainless steel chamber. After rough pumping at 1 X 10(-3) mbar with a mechanical pump to remove LN2, the chamber was evacuated with a cryopump or turbomolecular pump to achieve a hydrocarbon-free, ultra-high vacuum of 1 X 10(-8) mbar. Equilibrium temperature in the chamber before the drying cycle was -192 degrees C. The copper specimen block was equipped with a thermocouple and a programmable feedback-controlled heating circuit. Tissue was dried by increasing the specimen block temperature 1 degree C/hr during the critical drying phase while monitoring the rate of water removal with a partial pressure analyzer. Results obtained indicate that drying is complete below the devitrification temperature of amorphous phase tissue water. Dried tissue was fixed with osmium tetroxide vapor, vacuum-embedded in a low-viscosity epoxy resin, sectioned, stained, and viewed with the electron microscope. Processed tissue exhibits excellent morphological preservation without the use of pre-fixation or cryoprotective agents. Thin sections of this tissue are excellent for immunocytochemical staining and electron microprobe analysis.

Localization of transforming growth factor-beta 1 in mitochondria of murine heart and liver.

“…Although no single experiment reported herein inevitably leads to that conclusion, the totality of the data, obtained both by electron microscopy and cell fractionation of both heart and liver, using unique and different sets of analytical reagents for both sets of experiments, makes a compel- ling case that the results obtained are not artifactual. The metal mirror freezing technique, followed by slow and controlled molecular distillation of tissue water, is one of the best available methods for localization of diffusible macromolecules by electron microscopy (Linner et al, 1986). Likewise, cytochrome oxidase is an established marker for monitoring mitochondrial recovery during cell fractionation procedures and has been used for this purpose for many years (Hogeboom and Schneider, 1955 , although there is slight cross-reactivity with TGF-f3 (Flanders, unpublished data).…”

“…Electron microscopic immunohistochemistry of mouse and rat heart We have used the metal mirror freezing technique (Phillips and Boyne, 1984) for cryofixation, followed by high-vacuum molecular distillation for removal of tissue water, to prepare samples for immunoelectron microscopy (Linner et al, 1986). This method represents a fundamental advance in tissue preparation, making possible ultrastructural localization of soluble molecules without the problems of redistribution and loss that have limited the usefulness of conventional techniques.…”

“…Tissues were cryofixed on a life cell CV 100 cryofixation unit (kindly provided by LifeCell, The Woodlands, TX) according to a method described previously (Linner et al, 1986;Livesey et al, 1989). After freezing, the tissue blocks were transferred to Nunc tubes (Cryomed, Mt.…”

Electron microscopy of high pressure frozen samples: bridging the gap between cellular ultrastructure and atomic resolution

“…En bloc freeze drying of cryoWxed samples before resin embedding was shown to preserve the location of diVusible ions signiWcantly better than other methods (Edelmann 1986;Linner et al 1986). However, the structural preservation is mostly not as good as after freeze substitution (e.g., there is shrinkage of mitochondria).…”

The synthesis, kinetic characterization and application of biotinylated aminoacylchloromethanes for the detection of chymotrypsin and trypsin-like serine proteinases

“…For example, a biotinylated probe could be used to detect a proteinase blotted from a polyacrylamide gel by detection methods such as streptavidin/peroxidase (Guesdon & Avrames, 1979;Yolken et al, 1983) or streptavidin/alkaline phosphatase (O'Conner & Ashman, 1982). Or, in a similar vein, detection of proteinases on cells or in tissue sections could be achieved using streptavidin/ fluorescein (Childs & Unabid, 1982), streptavidin/Texas Red (Berger et al, 1982) for u.v.-microscopy or streptavidin/colloidal gold (Linner et al, 1986) for electron microscopy.…”