Self-pressurized rapid freezing (SPRF): a novel cryofixation method for specimen preparation in electron microscopy.

A method is described for the cryofixation of biological specimens for ultrastructural analysis and immunocytochemical detection studies. The method employs plunge freezing of specimens in a sealed capillary tube into a cryogen such as liquid propane or liquid nitrogen. Using this method a number of single-cell test specimens were well preserved. Also multicellular organisms, such as Caenorhabditis elegans, could be frozen adequately in low ionic strength media or even in water. The preservation of these unprotected specimens is comparable to that achieved with high-pressure freezing in the presence of cryoprotectant. The results are explained by the fact that cooling of water in a confined space below the melting point gives rise to pressure build-up, which may originate from the conversion of a fraction of the water content into low-density hexagonal ice and/or expansion of water during supercooling. Calculations indicate the pressure may be similar in magnitude to that applied in high-pressure freezing. Because the specimens are plunge cooled, suitable cryogens are not limited to liquid nitrogen. It is shown that a range of cryogens and cryogen temperatures can be used successfully. Because the pressure is generated inside the specimen holders as a result of the cooling rather than applied from an external source as in high-pressure freezing, the technique has been referred to as self-pressurized rapid freezing.

Steric hindrance in immunolabelling.

In this paper we describe immunocytochemical detection of PhoE pore protein in the outer membrane of E. coli K-12 cells in dependence of a variety of labelling approaches. Immuno-gold labelling on ultrathin cryosections showed a uniform, dense labelling of the outer membrane of all cells. However, using immunofluorescence, whole-mount or freeze-etch labelling methods, labelling was limited to less than 5% of the cell population. In order to understand this phenomenon, immunoincubated cells exhibiting less than 5% labelling were processed for cryo-ultramicrotomy. Reincubation of the sections with antibody and probe resulted in labelling of all of the cells. If an E. coli Gal-U mutant strain, defective in the lipopolysaccharide (LPS) carbohydrate chain length, was used, each approach rendered 100% labelling. From these results it is concluded that the antigenic sites of the PhoE pore protein are not accessible in intact 'wild-type' cells due to steric hindrance caused by the LPS carbohydrate chains. In cryosections this steric hindrance is partly precluded since antigenic determinants are exposed at the section surface in transversely cut membranes. Our results emphasize the necessity to compare results obtained by means of several, basically different approaches.

Controlled growth of colloidal gold particles and implications for labelling efficiency.

A new method is reported for the preparation of colloidal gold particles with diameters ranging between 5 and 12 nm. The initial gold particle population, with an average diameter of 5.6 +/- 0.9 nm, is prepared by reduction of chloroauric acid with white phosphorous. An increase in particle diameter by growth is obtained by reduction of chloroauric acid with white phosphorous in the presence of colloidal gold particles. The labelling efficiency of these gold particles, conjugated with protein A, in indirect immunolabelling experiments is investigated by labelling of beta-galactosidase on ultrathin cryosections of Escherichia coli cells. We demonstrate that the labelling efficiency is at least dependent on particle diameter, probe concentration and preparation method. In addition it is shown, that with this new method, gold particle populations can be prepared with minor overlap in diameter spreading. Therefore these gold probes are suitable for qualitative double labelling experiments. The quantitative aspect of immunolabelling is discussed.

Presynaptic localization of B-50 phosphoprotein: the (ACTH)-sensitive protein kinase substrate involved in rat brain polyphosphoinositide metabolism.

This study describes the ultrastructural localization in rat hippocampal tissue in situ and in isolated synaptosomes of the brain-specific phosphoprotein B-50, using affinity purified anti-B-50 immunoglobulins (IgGs). Evidence is presented for the presynaptic localization of B-50 in rat brain. Given this specific localization a model is presented outlining the presumed function of the B-50 protein in the membrane and describing possible neuromodulation by adrenocorticotropin hormone (ACTH)-like peptides.

Visualization of epidermal growth factor receptor in cryosections of cultured A431 cells by immuno-gold labeling.

Cryo-ultramicrotomy in combination with immuno-gold labeling has been demonstrated to present a powerful tool in the visualization of extra- and intracellular located antigens. We have applied this method to localize epidermal growth factor (EGF) receptor in cultured A431 human epidermoid carcinoma cells. However, both the labeling efficiency, maintenance of antigenicity, and the recognizability of the ultrastructure in cryosections are highly dependent upon the fixation procedures. Using 125I-EGF or a consecutive labeling with a monoclonal anti EGF-receptor antibody, rabbit-anti-mouse antibody and 125I-protein A, it was shown that maintenance of antigenicity was optimal using 2% paraformaldehyde as a fixative, whereas under these conditions also the recognizability of ultrastructure was sufficient. After appropriate fixation and labeling, gold particles were observed associated with various regions of the plasma membrane, including coated pits, and with various types of vesicles, including coated vesicles, intracellular vesicular membranes, multi-vesicular bodies and lysosomes. The results indicate that this method allows a visualization of EGF-receptors and resolution of the EGF-receptor processing pathway at the electron microscopic level, independent of the internalization process of labeled ligands.

Ultrastructural localization of S-antigen in retinal structures.

The localization of S-antigen, the soluble, uveitogenic, 51 kDa protein of the retina has been studied. Highly specific rabbit anti-bovine S-antigen antiserum reacted predominantly with the outer segment layer of the photoreceptor cells of rat retina in the indirect immunofluorescence technique, provided that the tissue was fixed by perfusion of the light adapted animal. More detailed information was obtained by immuno-electron microscopy using the same fixation technique and Protein A-coated gold particles for labeling. S-antigen was found to be predominantly bound to the rod outer segment plasma membranes and to the discs. The possible implication of this localization is discussed in view of the function of S-antigen.

Intracellular localization of lipoxygenases-1 and -2 in germinating soybean seeds by indirect labeling with protein a-colloidal gold complexes.

Soybean lipoxygenases-1 and -2 were localized intracellularly in seeds at various stages of germination by indirect labeling of cryosections with protein A-colloidal gold complexes. Two sizes of gold particles (Au(5) and Au(16)) were used in single- and double-labeling experiments. In primary leaves, lipoxygenases are demonstrated to occur in vacuolating parenchyma cells but not in massive, nondifferentiated cells. In cotyledons, both isoenzymes are localized in the cytoplasm of storage parenchyma cells and in an aberrant type of protein bodies, occurring in hypodermis and vascular bundle sheath cells. No association has been found with either protein bodies in storage parenchyma cells or lipid bodies, mitochondria, and other organelles in any type of cell. The possible significance of lipoxygenase in the metabolism of storage lipids and its possible function as a regulatory enzyme are discussed on the basis of the random distribution throughout the cytoplasm of storage parenchyma cells and the course of biochemical processes during seed germination.

Intracellular CBG-like molecules in the rat pituitary.

The localization of transcortin (CBG) in pituitary cells of the rat was investigated using the peroxydase-antiperoxydase (PAP) technique. A rabbit antiserum against purified rat plasma transcortin was used as the primary antiserum. Transcortin-like (CBG-like) immunoreactive products were found in the cytoplasma of certain cells in the anterior pituitary, but not in the intermediate lobe and weakly in the posterior pituitary. It is postulated that the CBG-like molecules participate in the cellular uptake process of corticosterone, thereby modulating the feedback signal of this steroid on pituitary function.

An evaluation of the cryosectioning of fixed and cryoprotected rat liver.

The influence of temperature and speed on cryosectioning was studied by replication of the surfaces of both the sections and the specimen blocks. At 90 mm/s and at temperatures lower than -70 degrees C, specimen block surfaces displayed fracture images similar to those encountered with standard freeze-fracturing procedures; but at a speed of 0.1 mm/s, fracture images were found only at temperatures lower than -120 degrees C. Replicas of both sides of cryosections never displayed fracture images. The discrepancy between the surface structure of cryosections and specimen blocks is discussed from the aspect of the preservation of ultrastructure of cells.

Immunocytochemistry of gonadotropic cells and identification of cell types in ultrathin cryosections of the pituitary of the rainbow trout, Salmo gairdneri.

The most frequently occurring cell types in the pars distalis of the pituitary gland of the rainbow trout, (i) the lactotropic, (ii) the gonadotropic, and (iii) the somatotropic cells, were identified in cryosections. Their morphological characteristics were compared with those of Epon-embedded material. Cell location, cell form, position of the nucleus, arrangement of rough endoplasmic reticulum and sizes of secretory granules proved to be useful parameters for identification. The size distribution of secretory granules of corresponding cells in cryosections and Epon sections proved to be similar. Additionally, both the immunoferritin and the unlabeled antibody enzyme method were applied for the immunocytochemical labeling of gonadotropic hormone-producing cells in cryosections. Anti-salmon-GTH as well as anti-carp-GTH serum showed the presence of GTH in both the smaller and the larger granules of the classical GTH cells, but also produced a reaction in TSH cells. Labelling of TSH cells was absent when using anti-beta-carp-GTH. Specificity of the reaction depended upon the degree of dilution of the anti-GTH serum. Results with dilutions of 1:4,000 and 1:8,000 in the unlabeled antibody enzyme method, and of 1:8,000 up to 1:32,000 in the immunoferritin technique were optimal. Acid phosphatase activity in the smaller granules was demonstrated by enzyme cytochemistry in Epon sections. The relationship of the presence of hormone in these granules is discussed. The high sensitivity of the immunocytochemical labeling procedure is discussed with respect to cryo-ultramicrotomy.

Modification of the Reichert FC-2 cryoattachment to improve ultrathin sectioning reproducibility.

Modifications of the Reichert FC-2 cryomicrotome attachment which facilitate sectioning of frozen material are described. Most valuable was the construction of a specimen holder chuck made of brass and connected to the specimen arm with a glass rod. Other changes include improved shielding from heat influx, a constant nitrogen pressure cooling system and a stable cryochamber mount. These modifications considerably improve reproducibility.

Bovine olfactory and nasal respiratory epithelium surfaces. High-voltage and scanning electron microscopy, and cryo-ultramicrotomy.

High-voltage transmission electron microscopy and cryo-ultramicrotomy together with scanning electron microscopy and some conventional transmission electron microscopy of ultrathin sections have been applied to the mucous surfaces of bovine olfactory and respiratory epithelia. Distal segments of olfactory cilia tend to run in parallel and could be followed over distances up to about 30 micrometer using high-voltage electron microscopy. This technique and scanning electron microscopy showed that on average 12--13 of such cilia could be observed per nerve ending. After correction for obscured cilia this number becomes about 17. High-voltage micrographs and micrographs made from sections prepared with a cryo-ultramicrotome showed the presence of electron-lucent pockets inside the olfactory mucus. The latter technique also showed that the mucus itself is not fibrous, but rather a continuum varying in electron density. The mucus layer contains various granular structures. Ciliary and microvillar membranes appear thicker with cryo-ultramicrotomy than when the sections are prepared with conventional techniques. The cores of the axonemal microtubules in olfactory as well as in respiratory cilia are darkly stained with this technique. Vesicles present inside the nerve endings are also darkly stained. Dimensions and some other numerical values of interest in olfaction are presented.