Improved preservation of intramitochondrial granules in rat-incisor odontoblasts by rapid-freezing and freeze-substitution fixation

Rat incisor tissue sections were fixed by a modified version of the malachite green-aldehyde method (MGA) composed of rapid-freezing, malachite green-acrolein staining, and osmium tetroxide freeze-substitution (Fr.MGAO). In the pre-dentin, a thick, dense network of branched fibrous structures was observed. Cryotechniques allowed visualization of complexes about twice as thick and dense as the aggregates visualized on MGA-treated sections. Pretreatment of rapid-frozen samples with methanol before freeze-substitution fixation and staining prevented staining of the complexes otherwise revealed by the Fr.MGAO method. Electron-dense material stained by this procedure resisted de-mineralization with EDTA, while intramitochondrial granules and dentin crystallites were dissolved. EDTA treatment demonstrated unequal distribution of Fr.MGAO staining in dentin in the form of tiny dots underlining the collagen fibers. These results support the concept that rapid-freezing, followed by staining and freeze-substitution fixation, improves preservation of the phospholipids visualized as extracellular matrix components in pre-dentin and dentin of rat incisors.

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Freeze-substitution of rye grass and ragweed pollen grains

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100160984 ◽

1990 ◽

Vol 48 (3) ◽

pp. 686-687

Author(s):

Liza B. Martinez ◽

Susan M. Wick

Keyword(s):

Cell Function ◽

Pollen Grains ◽

Freeze Substitution ◽

Cell Types ◽

Rapid Freezing ◽

Rye Grass ◽

Acrylic Resins ◽

Allergenic Proteins ◽

Cold Embedding ◽

Structural Preservation

Rapid freezing and freeze-substitution have been employed as alternatives to chemical fixation because of the improved structural preservation obtained in various cell types. This has been attributed to biomolecular immobilization derived from the extremely rapid arrest of cell function. These methods allow the elimination of conventionally used fixatives, which may have denaturing or “masking” effects on proteins. Thus, this makes them ideal techniques for immunocytochemistry, in which preservation of both ultrastructure and antigenicity are important. These procedures are also compatible with cold embedding acrylic resins which are known to increase sensitivity in immunolabelling.This study reveals how rapid freezing and freeze-substitution may prove to be useful in the study of the mobile allergenic proteins of rye grass and ragweed. Most studies have relied on the use of osmium tetroxide to achieve the necessary ultrastructural detail in pollen whereas those that omitted it have had to contend with poor overall preservation.

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Ultrastructural and immunocytochemical studies of the rat hypothalamo-neurohypophysial system processed by rapid freezing and freeze-substitution fixation

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100161989 ◽

1990 ◽

Vol 48 (3) ◽

pp. 884-885

Author(s):

Seiji Shioda ◽

Yasumitsu Nakai ◽

Atsushi Ichikawa ◽

Hidehiko Ochiai ◽

Nobuko Naito

Keyword(s):

Osmium Tetroxide ◽

Freeze Substitution ◽

Ultrastructural Localization ◽

Wistar Rat ◽

Neurosecretory Cells ◽

Rapid Freezing ◽

Sodium Metaperiodate ◽

Tissue Sections ◽

Ultrathin Sections ◽

Electron Microscopes

The ultrastructure of neurosecretory cells and glia cells in the supraoptic nucleus (SON) of the hypothalamus and the neurohypophysis (PN) was studied after rapid freezing followed by substituion fixation. Also, the ultrastructural localization of vasopressin (VP) or its carrier protein neurophys in II (NPII) in the SON and PN was demonstrated by using a post-embedding immunoco1loidal gold staining method on the tissue sections processed by rapid freezing and freeze-substitution fixation.Adult male Wistar rat hypothalamus and pituitary gland were quenched by smashing against a copper block surface precooled with liquid helium and freeze-substituted in 3% osmium tetroxide-acetone solutions kept at -80°C for 36-48h. After substituion fixation, the tissue blocks were warmed up to room temperature, washed in acetone and then embedded in an Epon-Araldite mixture. Ultrathin sections mounted on 200 mesh nickel grids were immersed in saturated sodium metaperiodate and then incubated in each of the following solutions: 1 % egg albumin in phosphate buffer, VP or NPII (1/1000-1/5000) antiserum 24h at 4°C, 3) colloidal gold solution (1/20) 1h at 20°C. The sections were washed with distilled waterand dried, then stained with uranylacetate and lead citrate and examined with Hitachi HU-12A and H-800 electron microscopes.

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