Gap junction structures. VIII. Membrane cross-sections.

Profiles of negatively stained gap junctions have been measured by grid sectioning. After normal levels of electron irradiation, the membrane thickness shrinks to about half that of unirradiated controls, but no shrinkage occurs in the hexagonal lattice plane. Even under low irradiation conditions, there is significant thinning of the membranes. Edge views, in which rows of connexons are aligned parallel to the beam, were obtained from grid sections, folds in normal negatively stained specimens, and sections of a positively stained specimen. Averaging these micrographs with the translational and mirror symmetry of the projected lattice image displays conserved and variable features in the stain distribution of different specimens. Variations in the relative amount of negative stain in the gap at the surfaces and in the channel are uncorrelated with the irradiation but appear to depend on the local staining conditions and the integrity of the connexons. The dimensions measured from previously unirradiated grid sections, folds, and positively stained sections are in accord with x-ray diffraction measurements. Radiation-induced shrinkage can be accounted for by mass loss principally from the membrane bilayer. Disordering of the surface structure appears to be correlated with the radiation sensitivity of the bilayer; in contrast, the gap structure is well preserved under a variety of conditions.     

Vitreous Carbon: A New Material for Making Microtome Knives

The quality of sections obtained by microtomy depends to a large extent on the quality and characteristics of the microtome knife itself. Despite the need for improved microtomy techniques, there have been few significant developments since the introduction of glass and diamond knives in the 1950's. The manufacture of microtome knives from vitreous carbon provides new possibilities for developing both improved methods and improved equipment for specimen sectioning. Vitreous carbon has unique physical properties that lend themselves to the generation of precision cutting edges. Such an edge can be obtained either by breaking a piece of vitreous carbon or by using lapidary techniques. The resultant edge seems well adapted to both thick and thin sectioning. The introduction of vitreous carbon as a sectioning tool offers a significant alternative to metal, glass and diamond knives.     

A new technique for hard tissues

Fossil tissues generally require specialized processing. Most thin sectioning techniques yield unstained sections or require unwieldy methods to produce stained sections. I outline here two simple techniques for producing stained, ground, thin sections using readily available Romanowsky-type cytology stains and a urine sediment stain. Results are comparable to hematoxylin and eosin stained specimens.     

Structure of molluscan thick filaments: a common origin for diverse appearances.

Thick filaments from the smooth adductor muscles of the oysters Ostrea edulis and Crassostrea angulata have been examined in the electron microscope after negative staining. The two well-known patterns of stain (whose origin and relation have been uncertain), one a series of transverse narrow lines at intervals of 144 Å along the filament axis and the other a regular two-dimensional arrangement of stained spots (Bear &; Selby, 1956), are found to be mutually interconvertible by rotating the grid around the filament axis. This is interpreted to mean that the spots are the projections of stained regions running through the filament in a common direction. Only when looking along this direction will the net pattern be seen with maximum clarity and sharpness. On rotation of the filament round its axis, the spots broaden transversely to the axis, overlapping and ultimately only the axial periodicity will remain. The structure is therefore not helical, but resembles a crystal lattice, although no period can be discerned normal to the net plane.The addition of 10 mm-EDTA to all solutions used in the filament preparation (except the stain), especially when ammonium molybdate is the stain employed, removes many puzzling appearances (probably caused by positive staining) which render the interpretation difficult. The appearance of the negatively stained filament can be related to the stain patterns in negatively stained paramyosin paracrystals (Cohen et al., 1971).     

The pleated sheet: an unusual negative-staining method for transmission electron microscopy of biological macromolecules

A novel method for preparing negatively stained specimens is described which appears to improve the routine resolution of biological structure in direct images obtained by transmission electron microscopy. In the new method, which we term the pleated sheet technique, macromolecules are adsorbed to a carbon film by the Valentine procedure (R. Valentine, B. Shapiro, and E. Stadtman (1968) Biochemistry, 7, 2143-2152), and the film then carefully pleated while in contact with a 1% uranyl formate solution to trap stain within the folds of pleats. A grid is placed on the compressed film, and film plus grid retrieved with a Saran Wrap drum. Subsequent dehydration produces a filmed grid containing negatively stained macromolecules within the folds of pleated regions and positively stained macromolecules in single sheet regions. The effect of sandwiching sample and stain between carbon layers is to produce exceedingly uniform negative staining so that stain contours more accurately and more reproducibly reflect true molecular contours. Electron micrographs of IgG and IgA molecules prepared by these methods are exhibited that permit unambiguous comparison of structure imaged in the electron microscope against known structures solved by single-crystal X-ray diffraction. Correlation is excellent; the smallest resolvable element in micrographs is an immunoglobulin domain, whose molecular weight is 12 000.     

Block Staining of Botanical Materials

Plant materials, including coleus stem tips, Psilotum stems and onion root tips, were stained in iron-mordanted celestine blue and safranin (Gray and Pickle 1956) prior to embedding and sectioning. Mitotic figures as well as general morphological and anatomical features are adequately stained by this procedure. The plant materials, subdivided so that the largest dimension is about 8-10 mm, are stained for 12-48 hr. The time is dependent upon the tissue and dilution of the stain used. Excess stain is washed out and the tissues are dehydrated, embedded, sectioned and mounted on slides in the usual manner. Following removal of the wax by xylene the sections may be counterstained, or a cover slip may be added immediately.     

Applying methods of hard tissues preparation for wood anatomy: Imaging polished samples embedded in polymethylmethacrylate

Cambial activity records short and long-term environmental signals in xylem anatomy, creating a permanent archive. Quantitative wood anatomy deciphers the relationship between cell structure and function in a spatiotemporal context. Obtaining high-resolution images of wood anatomical preparations is a critical stage in the process of decoding this information. Damage to cellular structures when sectioning by microtome is one of the main problems in the preparation of high-quality micro-sections. Cell damage leads to the occurrence of artifacts – most often related to broken cell walls – hindering the performance of image recognition programs, and increasing the time spent on the manual editing of images. In this work, we propose an alternative method to microtomy, based on embedding-polishing protocols established for hard tissue preparation. Wood samples are embedded in a transparent and non-reactive resin as polymethylmethacrylate (PMM) that is subsequently ground and polished. Being able to acquire images from the stained or unstained polished surfaces of the PMM-blocks and sections (thinner than 100 μm) by using a wide range of optical methods such as reflected polarizing microscopy, epifluorescence microscopy, bright-field microscopy with diffuse illumination and circularly polarizing microscopy. This embedding method improves the mechanical integrity and quality of wood anatomical preparations, eliminating the problem of broken cell walls. Furthermore, this technique allows the preparation and analysis of large tissue surfaces.     

Negative-staining autoradiography: a new technique for ultracryotomy utilizing an interposed film

A new radiocytochemical technique is reported for ultrastructural localization of diffusible substances, using negatively stained ultra-cryostat sections. A sheet of film interposed between the cryostat section and the emulsion layer has rendered negative-staining autoradiography (NSA) practical. The rationale of NSA is that the film completely shields the section from all moisture-producing autoradiographic processes, so that phosphotungstic acid (PTA) can stain the section either before or after autoradiography (ARG), without the possibility of ultrastructural damage by alkaline solutions, interference between PTA and photoprocessing compounds, and superimposed images of a gelatin layer stained with PTA. As a model to demonstrate the newly developed procedure of NSA, rat brains were labeled with [125I]-triiodothyronine, fixed with tannic fixative, immersed in a cryoprotectant, frozen in liquefied propane, and cryostat sectioned. The resulting higher yield of radioactivity (85%) on the section was confirmed by a radiation counter. The retention rate was approximately 20% greater than that of conventional sections. Developed silver grains were found on synaptic vesicles and mitochondria in the polymorphic layer of the dentate gyrus. In this report we will also discuss the problems associated with cryostat sectioning of fresh tissues, the concept of ARG resolution, the distribution pattern of developed silver grains, and the possible applications of NSA.     

Analysis of Distributed Growth of Saccharomyces cerevisiae Cells Immobilized in Polyacrylamide Gel

A technique is described for the quantitative determination of the distributed growth of Saccharomyces cerevisiae immobilized in polyacrylamide gel. Gel specimens were embedded in paraffin or gelatin and paraffin before sectioning and staining. Photomicrographs of specimen sections were enlarged, and cell microcolony volumes were determined as a function of position in the gel by grid transparency analysis. Overall cell densities within the gel were calculated for a quantitative comparison with values measured by a second spectrophotometric method. The results show good agreement and demonstrate the sigmoidal growth of the immobilized cells, reaching a maximum steady-state value. The technique shows promise as a general method for following the transient growth of organisms immobilized within gel particles.     

Negative staining studied by TEM and STEM mass measurements: Preliminary observations on collagen sheets negatively stained with uranyl acetate

Quantitative mass measurements by dark-field scanning transmission EM and conventional bright-field transmission EM have been used to determine the increase in mass brought about by negative staining. pN-collagen (which forms sheets of uniform thickness and known mass per unit area) was used as a test specimen; the negative stain was uranyl acetate (1%, pH 4.4). The mass increase corresponded to the addition of roughly 8 uranyl acetate molecules per nm² for lightly negatively stained specimens; for heavily stained specimens, it was 30 molecules or more. The appearance of the image was related to the mass increase. This preliminary study shows that mass measurements can provide a basis for the quantitative interpretation of images from negatively stained specimens.     

Equivalence of the projected structure of thin catalase crystals preserved for electron microscopy by negative stain,glucose or embedding in the presence of tannic acid

Thin crystals of beef liver catalase have been examined by electron microscopy following various preservation procedures. In the first part of this investigation, micrographs of three principal projections were obtained from thin sections of micro-crystals embedded in the presence of tannic acid. Computer reconstructions confirmed the space group assignment of P2₁2₁2₁ and permitted the packing arrangement of the catalase tetramers to be deduced to a resolution of about 20 Å. These results corroborate the packing model for this crystal form proposed by Unwin (1975) on the basis of molecular modeling of one projection. In the second part of this investigation, the projected structures of the thin crystals in various preserving media were compared. The negative contrasting of crystals embedded in the presence of tannic acid was confirmed by direct comparison with nonembedded, negatively stained thin platelet crystals. In addition, good agreement at 20 Å resolution was observed between the structure of negatively stained crystals and the structure of crystal platelets preserved in glucose and examined by lowdose methods, while moderate agreement was established with the published data of Taylor (1978) for crystals embedded in thin ice films. Tannic acid alone was also found to serve as a suitable medium for preserving catalase crystals to a resolution of 3.7 Å as judged by electron diffraction. Overall, we demonstrate that projections obtained from thin sections of catalase crystals embedded in the presence of tannic acid can provide a reliable, negatively contrasted representation of the protein structure to 20 Å resolution. Examination of sectioned crystals could thus provide a useful adjunct to X-ray crystallographic studies of protein crystals and three-dimensional reconstruction of crystal thin sections should ultimately be possible.     

A two-color acid-free cartilage and bone stain for zebrafish larvae

Traditionally, cartilage is stained by alcian blue using acidic conditions to differentiate tissue staining. The acidic conditions are problematic when one wishes to stain the same specimen for mineralized bone with alizarin red, because acid demineralizes bone, which negatively affects bone staining. We have developed an acid-free method to stain cartilage and bone simultaneously in zebrafish larvae. This method has the additional advantage that PCR genotyping of stained specimens is possible.     

Direct visualization of microtomy artefacts in sections of twisted fibrous extracellular matrices

Twisted fibrous extracellular matrices observed in section often show alternating clear and dark bands. Three different methods of observation (high voltage electron microscopy, shadowing of thin sections and stereoscopic views) show the presence of ruffling effects and relief at the surface of crab cuticle sections. These effects appear uneven on both sides of the sections. As shown in a series of diagrams, the localization of the microtomy artefact is a function of the orientation of the cuticle laminae relative to the knife direction, and this creates variations in the position and the extent of the microtomy effect over each lamina. Confirmation of this analysis is obtained in a particular geometrical situation which appears in sections of tubercles in the crab cuticle where the twisted plywood stratification is deformed into a dome. By shadowing thin sections, perpendicular to the tubercle axis, nested crescents are visualized on the surface of the samples. All observations demonstrate that the clear and dark lamellae are due to a microtomy artefact which is a three-dimensional process, and not, as usually considered, due to chemical or physical variations in the structure.     

Tape transfer sectioning of tissue microarrays introduces nonspecific immunohistochemical staining artifacts

Abstract

Tissue microarrays place tens to hundreds of formalin fixed, paraffin embedded tissue cores into a paraffin block in a systematic grid pattern that permits their simultaneous evaluation in a single section. The fragmented nature of the tissue cores often makes sectioning of tissue microarrays difficult so that the resulting disks of tissue lose their shape, fracture or fall out of the paraffin section altogether. We have evaluated an alternative sectioning protocol for stabilizing the tissue microarray surface by placing an adhesive tape “window” over the face of the paraffin block prior to sectioning. Once sectioned, the tape/sections are transferred directly onto coated microscope slides, thereby avoiding routine floating of sections on a water bath. After sectioning with either the tape transfer or standard protocols, slides were stained either using hematoxylin and eosin or immunohistochemistry using antibodies to S-100 protein and the tissue specific antigens, keratin (AE1/3) and the leukocyte common antigen CD45. We found that the tape method produced thicker sections that were darker and more densely packed with loss of tissue definition compared to sections prepared using water bath flotation. Quantitative image analysis of immunohistochemical staining demonstrated that the tape method produced a higher incidence of nonspecific staining, which raised the potential for false positive staining.     

Untersuchungen zur Anfälligkeit und Resistenz von Kopfsalat (Lactuca sativa) gegen falschen Mehltau (Bremia lactucae) III. Peroxydase-, peroxydatische Katalase-und Polyphenoloxydase-Aktivitäten

Investigations on the susceptibility of head lettuce (Lactuca sativa) to downy mildew (Bremia lactucae) III. Activities of peroxidase, catalase and polyphenoloxidase Host cell walls in contact with intercellular hyphae of Bremia lactucae stain electron positively in susceptible and incompletely resistant varieties of lettuce after appropriate electron microscopy preparation for peroxidase activity. The outer membranes of the mitochondria of the parasite also stained darkly in susceptible varieties whereas in incompletely resistant plants Bremia innermost mitochondrial membranes and host cell mitochondria were darkly stained. This latter observation suggests increased respiration and could be explained as a resistance reaction. Catalase activity was observed in the microbodies of susceptible, in incompletely resistant and healthy varieties. There were no differences in stain intensity in the three kinds of varieties suggesting that catalase activity is not involved in resistance reactions. Polyphenoloxidase activity was infrequently observed on the host cell wall in susceptible and healthy plants, whereas strong activity in incompletely resistant varieties was observed in vesicles in the haustorial sheath. These vesicles were not surrounded by unit membranes and therefore could not have originated from the unit membranes of the extrahaustorial matrix or from the host plasmalemma. They may have been derived from the host protoplast and involved in inactivation of parasite produced toxins thereby contributing to resistance.     

Gap junction structures. VII. Analysis of connexon images obtained with cationic and anionic negative stains

Micrographs of isolated gap junction specimens, negatively stained with one molybdate, three tungstate and three uranyl stains, were recorded at low and high irradiation. Fourier-averaged images of the negatively stained gap junctions have been self-consistently scaled to identify conserved and variable features. Intrinsic features in the hexagonally averaged images have been distinguished from residual noise by statistical comparisons among similarly prepared specimens. The cationic uranyl stains can penetrate the axial connexon channel, whereas the anionic stains are largely excluded; these observations indicate that the channel is negatively charged. Variability in the extent of the axial stain penetration, and enhancement of this staining by radiation damage and heating may be accounted for by a leaky, labile channel gate. The peripheral stain concentrations marking the perimeter of the skewed, six-lobed connexon image and the stain-excluding region at the 3-fold axis of the lattice, which are seen only under conditions of low irradiation with both anionic and cationic stains, are identified as intrinsic features of the isolated gap junction structure. The stain concentrations located approximately 30 A from the connexon center appear to be symmetrically related on opposite sides of the junction by non-crystallographic 2-fold axes oriented approximately 8 degrees to the lattice axes at the plane of the gap. The radiation-sensitive hexagonal features seen in the negatively stained images may correspond to substructure on the cytoplasmic surfaces of the paired gap junction membranes.     

A solvent-free coating-procedure for the improved preparation of cryostat sections in light microscope histochemistry.

A new technique is presented for the external stabilization of cryostat sections by spraying the specimen surfaces with an aqueous solution of poly(vinyl alcohol) before each sectioning stroke. The spray freezes upon the surface and forms a tough coating which facilitates subsequent sectioning and handling especially of difficult material. The sections are affixed upon cold glass slides covered with an improved formulation of pressure-sensitive adhesive. During further processing of the affixed sections, the PVA-coating and any surrounding supporting medium dissolve without traces in the first aqueous incubation or staining solution.     

Technical advance: an aniline blue staining procedure for confocal microscopy and 3D imaging of normal and perturbed cellular phenotypes in mature Arabidopsis embryos

A new method is described for fluorescent imaging of mature Arabidopsis embryos that enables their cellular architecture to be visualized without the need for histological sectioning. Mature embryos are stained with aniline blue and cleared with chloral hydrate to allow high-resolution confocal imaging of individual cells within the embryo prior to germination. The technique allows the collection of longitudinal optical sections throughout the cotyledon, hypocotyl and root of wild-type Arabidopsis C24 embryos. Every cell within the mature embryo can be visualized with sufficient clarity and resolution to allow three-dimensional analysis of cellular architecture. Optical sectioning of mutant gnom, short-root and scarecrow embryos, and through root meristems disrupted as a consequence of targeted misexpression of diphtheria toxin, demonstrate the potential of this technique for visualizing the cellular organization of mutant and perturbed embryonic phenotypes.     

Two-dimensional crystals of a membrane protein: arrangement of subunits within the crystal sheet

Two-dimensional crystals have been prepared from the photosynthetic reaction center of Rhodopseudomonas viridis. Filtered images of these crystals show individual subunits approximately 4.5 nm in diameter arranged at a center-to-center distance of 6.4 nm. Our previous studies suggested that each subunit within such a sheet corresponds to a single photosynthetic reaction center. Air-dried and freeze-etched shadowed preparations of the crystals yield images which are quite different from negatively stained material. Rotary-shadowed surfaces of the crystals show rows of wedge-shaped particles separated by 3 nm furrows. Two such wedge-shaped particles occupy the 12.1 X 12.9 nm area in which four negatively stained subunits are normally visualized. Close analysis of these shadowed pictures suggests that both the shadowed and negatively stained images can be accounted for by a single model of subunit arrangement within the crystal. Within each 12.1 X 12.9 nm unit cell, two subunits are placed near one surface of the sheet, and two others are near the other surface. All four subunits are visible in negative stain. When the surface is shadowed, only the two subunits which project above the surface of the sheet accumulate appreciable amounts of the heavy metal shadow. Because of their close position, one subunit shades the other, forming the wedge-shaped appearance characteristic of the crystal. The only arrangement consistent with both shadowed and negatively stained images is one in which the two raised subunits occupy positions at either end of a diagonal across the unit cell. The analysis of shadowed images indicates that the plane group of the crystals is P22(1)2(1).     

Chlorantine fast green BLL as a stain for callose in oak phloem

Sections of oak bark were stained with chlorantine fast green BLL, used as a 0.25% aqueous solution. All tissues were unstained, except for local deposits of material associated with phloem cell walls, which stained deep green. This green-staining material also stained specifically with resorcinol blue and with the aniline blue fluorescence technique, the usual histochemical tests for callose. The chlorantine fast green-staining material was removed from sections by treatment with a beta-1,3-glucan hydrolase. It is concluded that chlorantine fast green BLL stains callose in plant sections and is a useful additional stain for the histochemical detection of this polymer.