A technique for revealing tissue embedded within resin blocks

A technique is described for revealing in fine detail large pieces of tissue still embedded within rough, scratched or partly-trimmed blocks of epoxy and other resin before sectioning. The pieces of tissue immersed in baths of oil or glycerol and illuminated by an appropriate optical system, may be photographed to provide a guide to subsequent sectioning, aiding the precise location of elements in the sections back to their position in the parent tissue, and for tracing the connections of the element between serial sections. The method has been used by the authors in sectioning for light microscopy, but could equally be used with sections for electron microscopy.     

A new method for comparative light and electron microscopic studies of individual cells,selected in the living state

A method is described which permits comparative light and electronmicroscopic studies of cell cultures, cell spreads or single selected cells which have been kept in the Plastic Film Dish (PFD). The PFD is a versatile large surface tissue culture chamber which, for electron microscopy, is mounted with a transparent FEP-Teflon film bottom. Cells are observed, selected and marked on the PFD-bottom with a high power inverted light microscope. The cells are fixed and dehydrated with a semi-automatic device while they are still in situ in the PFD. During the preparation steps for electron microscopy the topographical relationship between individual cells and between cells and cell support is accurately retained. After embedding and polymerization the Teflon film is easily peeled off the polymerized Epon, leaving a replica of the mark around the selected cell. This permits relocation of the selected cells for ultrathin sectioning in a plane plan-parallel to the original cell support. To enable orientated sectioning of selected cells in a plane perpendicular to the cell support, cells are tagged with Letraset-letters after original embedding and polymerization. Subsequently the re-embedded polymerized specimens are orientated in the microtome in a position which permits controlled thin sectioning of the tagged cells in the previously selected plane.     

A method of embedding tissue culture preparations in situ for transmission electron microscopy

A method for embedding tissue culture preparations, grown on Millipore filters, in Araldite is described. Accurate light microscopic localization of areas suitable for ultrathin sectioning is possible. The plane of sectioning relative to the Millipore filter can be varied to suit individual experimental needs.     

Why low temperature embedding for X-ray microanalytical investigations?

Freeze-drying followed by infiltration with resin and polymerization by UV light at low temperatures and under constant vacuum conditions is an alternative tissue preparation technique for microprobe analysis. Embedding is carried out with the nonpolar low-temperature embedding resin (Lowicryl HM20) which allows infiltration and polymerization at temperatures down to ?50°C. Sections of low temperature embedded material can be cut dry at ?60°C or at room temperature. Sectioning at low temperatures is an alternative for preparations that are difficult to cut at room temperature. The morphological preservation is adequate for the identification of structures such as mitochondria, lysosomes and different types of endoplasmic reticulum in liver cells. Some physical properties of Lowicryl resins, such as mass loss under the electron beam and high contrast, are positive characteristics for the analysis of semi-thick sections. No significant differences in the elemental composition could be detected between tissue which was freeze-dried or freeze-substituted prior to embedding. Freeze-drying is less time consuming. By avoiding contact with organic solvents the risks of ion loss and redistribution are diminished. In contrast to freeze-dried thin cryosections, low temperature embedded material can be sectioned for light microscopy and areas of interest chosen for further thin sectioning. This is of great importance in work with tissues with complicated morphology and heterogeneous cell populations. The initial preparative step—the cryofixation— determines to a high degree the morphological preservation of freeze-dried and embedded tissue.     

Alteration of cartilage matrix morphology with histological processing

An interlacunar network in the extracellular matrix of femoral head articular cartilage of neontal rats was seen by light microscopy to: (1) consist of elements, 0·5 μm thick, which occurred as individual elements, as bundles of elements, and as fused elements, (2) stain intensely with toluidine blue, methylene blue, and safranin O, and (3) connect chondrocytes by inserting on the chondrocyte capsules which were composed of morphologically and cytochemically similar material. By electron microscopy, the single elements were seen to be composed of thicker, denser staining areas of the honeycomb appearing matrix and the fused elements appeared as non-membrane bound channels containing granular material. Articular cartilage was processed using combinations of fixatives, dehydrating agents, and embedding media. Regardless of fixation, demineralization, or embedding, the network was not seen after dehydration of the cartilage with methanol, ethanol, acetone or tert-butanol but was seen after dehydration with aqueous solutions of glycol methacrylate, propylene oxide, 2-propanol or 2,2-dimethoxypropane. Network visualization following a variety of methods demonstrated that no single fixative, dehydrating agent, or embedding medium caused its formation. The presence of the network in different cartilage zones, its consistent morphology by light and electron microscopy, the uniformity of the elements in their connection with the chondrocytes, and presence in fresh-frozen sections suggest the network may be real, but rigorous evidence for its existence in vivo is still required. Since cartilage morphology was altered by histological methods, especially dehydration, common methods used in studying connective tissue matrix should be evaluated to determine their effect on matrix morphology.     

Capsule-supporting ring: a new device for resin embedding of glass-mounted specimens

The goal of specimen preparation for transmission electron microscopy is to obtain high-quality ultra-thin sections with which we can correlate cellular structure to physiological function. In this study, we newly developed a capsule-supporting ring that can be useful for resin embedding of glass-mounted specimens. The present device allowed us to re-embed a semi-thin section on a microscope slide into a resin block not only for efficient ultra-thin sectioning but also for a correlative light and electron microscopy. Similar to epoxy resins for morphological observations, semi-thin sections of low-viscosity hydrophilic resins, such as Lowicryl series, can be re-embedded into the resin, which can be useful for cytochemical gold labelling. A further application of the present device improved flat embedding of cultured cells on glass cover slips for electron microscopy, preserving in situ sub-cellular structures close to their native state. We practically describe the use of capsule-supporting ring and demonstrate representative micrographs as results.     

Rapid combined light and electron microscopy on large frozen biological samples

The use of large unfixed frozen tissue samples (10 × 10 × 5 mm³) for combined light microscopy (LM) and electron microscopy (EM) is described. First, cryostat sections are applied for various LM histochemical approaches including in situ hybridization, immunohistochemistry and metabolic mapping (enzyme histochemistry). When EM inspection is needed, the tissue blocks that were used for cryostat sectioning and are stored at −80 °C, are then fixed at 4 °C with glutaraldehyde/paraformaldehyde and prepared for EM according to standard procedures. Ultrastructurally, most morphological aspects of normal and pathological tissue are retained whereas cryostat sectioning at −25 °C does not have serious damaging effects on the ultrastructure. This approach allows simple and rapid combined LM and EM of relatively large tissue specimens with acceptable ultrastructure. Its use is demonstrated with the elucidation of transdifferentiated mouse stromal elements in human pancreatic adenocarcinoma explants grown subcutaneously in nude mice. Combined LM and EM analysis revealed that these elements resemble cartilage showing enchondral mineralization and aberrant muscle fibres with characteristics of skeletal muscle cells.     

A simple method for the handling and orientation of small specimens for electron microscopy

Cyanoacrylic glue (Eastman 910) was used to affix small pieces of nasal scrapings to lens paper immediately before fixation in the glutaraldehyde. The lens paper not only served to hold specimens together so that they were not lost during tissue processing, but also functioned as a ‘landmark’ for the specimens, so that specimens could be oriented in a specific manner during embedding and subsequent sectioning.     

Method for preparing thin sections of untreated equine hoof horn for electron microscopic examination

The preparation of hard tissues such as the equine hoof horn for electron microscopic examination is very difficult. In particular the penetration of fixatives and chemicals used during fixation and embedding is a problem. The objective of this study was to find and implement an alternative method enabling the preparation of high-quality thin sections of hoof horn and other hard tissue, which maintains the hard tissue ultrastructure and can be used for immuno-labeling. Compared to commonly used fixation and embedding techniques, the preparation of thin sections from untreated material method saves time and material and provides equivalent ultrastructural information. Furthermore, thin sections from untreated material are significantly larger and more homogeneous, more resistant to the electron ray, as well as more suitable for sectioning. The electron microscopical pictures obtained allow a comparison to previous test results achieved with fixed and embedded material. Using the preparation of thin sections from untreated material method, fixation and embedding artifacts are avoided, providing a clearer interpretation of the electron microscopical findings. Considerable advantages are achieved by using immunohistochemical techniques with untreated horn specimens because fixation invariably decreases antigenicity.     

A simple handling technique for mammalian oocytes and embryos during preparation for transmission electron microscopy

Due to their small size, mammalian oocytes and embryos pose unique problems during preparation for transmission electron microscopy. This paper outlines a method which combines protein embedding with centrifugation to locate the specimens on the face of a Beem capsule mould. This method facilitates both the processing of oocytes with minimal loss and rapid location of the specimens within the block for simultaneous sectioning, staining and examination.     

Comparison of different methods for thin section EM analysis of Mycobacterium smegmatis

Bacteria are generally difficult specimens to prepare for conventional resin section electron microscopy and mycobacteria, with their thick and complex cell envelope layers being especially prone to artefacts. Here we made a systematic comparison of different methods for preparing Mycobacterium smegmatis for thin section electron microscopy analysis. These methods were: (1) conventional preparation by fixatives and epoxy resins at ambient temperature. (2) Tokuyasu cryo-section of chemically fixed bacteria. (3) rapid freezing followed by freeze substitution and embedding in epoxy resin at room temperature or (4) combined with Lowicryl HM20 embedding and ultraviolet (UV) polymerization at low temperature and (5) CEMOVIS, or cryo electron microscopy of vitreous sections. The best preservation of bacteria was obtained with the cryo electron microscopy of vitreous sections method, as expected, especially with respect to the preservation of the cell envelope and lipid bodies. By comparison with cryo electron microscopy of vitreous sections both the conventional and Tokuyasu methods produced different, undesirable artefacts. The two different types of freeze-substitution protocols showed variable preservation of the cell envelope but gave acceptable preservation of the cytoplasm, but not lipid bodies, and bacterial DNA. In conclusion although cryo electron microscopy of vitreous sections must be considered the 'gold standard' among sectioning methods for electron microscopy, because it avoids solvents and stains, the use of optimally prepared freeze substitution also offers some advantages for ultrastructural analysis of bacteria.     

A reliable method of preparing undecalcified human bone biopsies for electron microscopic investigation

Undecalcified preparation of bone tissue for electron microscopic investigation depends on an adequate embedding method. A low viscosity resin proved to comply with the particular conditions as there is good infiltration of specimen by the monomer medium and also sufficient hardness of the polymerization product. Both properties stabilize the transition of soft tissue to mineralized bone surface; thus reproducible results of good quality are obtained in ultra-thin sectioning. A special preparation technique of human bone biopsies for simultaneous light and electron microscopic investigation has been developed. The method is based on experiences with approximately 300 iliac crest biopsies in metabolic and endocrine bone disorders.     

Freeze substitution followed by low melting point wax embedding preserves histomorphology and allows protein and mRNA localization techniques

Fixation and embedding are major steps in tissue preservation for histological analysis. However, conventional fixatives like aldehyde‐based solutions usually mask tissular epitopes preventing their immunolocalization. Alternative fixation methods used to avoid this drawback, such as cryopreservation, alcohol‐ or zinc salts‐based fixatives do not efficiently preserve tissue and cell morphology. Likewise, paraffin and resin embedding, commonly used for thin sectioning, frequently damage epitopes due to the clearing agents and high temperatures needed along the embedding procedure. Alternatives like cryosectioning avoid the embedding steps but yield sections of poorer quality and are not suitable for all kinds of samples. To overcome these handicaps, we have developed a method that preserves histoarchitecture as well as tissue antigenic properties. This method, which we have named CryoWax, involves freeze substitution of the samples in isopentane and methanol, followed by embedding in low melting point polyester wax. CryoWax has proven efficient in obtaining thin sections of embryos and adult tissues from different species, including amphioxus, zebrafish, and mouse. CryoWax sections displayed optimal preservation of tissue morphology and were successfully immunostained for fixation‐ and temperature‐sensitive antigens. Furthermore, CryoWax has been tested for in situ hybridization application, obtaining positive results. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

The preparation of cryosections from plant tissue: An alternative method appropriate for secondary ion mass spectrometry studies of nutrient tracers and trace metals

A method involving cryostat sectioning (10 μm thickness) and freeze-drying is presented for the preparation of plant tissue for microanalytical studies. The method is well suited for semi-quantitative imaging by secondary ion mass spectrometry (SIMS) and offers significant advantages over bulk freeze-dried or freeze-substitution preparations. Segments of corn or soybean root (5 mm) are quench-frozen, embedded externally, sectioned in a cryostat (10 μm), pressed onto ultrapure Si and slowly freeze-dried. Images of these sections with secondary electron microscopy and SIMS indicated good morphological preservation. It was possible to section tissues of a wide developmental range, as well as roots varying sixfold in diameter. SIMS images are presented which demonstrate the ability to detect and localize nutrient tracers, such as Rb⁺, following brief exposures (10 min) to the intact plant. Likewise, a toxic metal (Al) was localized in root tissue after brief exposure (<1 day) of the intact plant root to micromolar external concentrations. Elemental redistribution during processing was minimal, as demonstrated most explicitly by the lack of movement of loosely bound Ca from the outer cell walls into the adjacent embedding material. Preservation of compositional differences between cellular content and cell wall was supported by a semi-quantitative treatment of SIMS images.     

Cryoultramicrotomy versus plastic embedding: comparative immunocytochemistry of rat anterior pituitary cells

The anterior pituitary of the rat is used as a model for the study of the effects of freezing or plastic embedding on the maintenance of antigenicity. Rat anterior pituitaries are fixed in 2.5% glutaraldehyde in 0.1 m phosphate buffer pH 7.4. Some of the blocks are post-fixed before being divided into two lots. One batch is frozen, while the other is dehydrated and embedded. The indirect antibody enzyme method is applied to ultrathin sections obtained by cryoultramicrotomy after freezing or by sectioning after embedding. All six pituitary hormones are detected by both methods. Comparison shows that the morphological characteristics are identical for both techniques, though ultrastructural preservation is better after embedding. Immunoreactivity is found in secretory granules and sometimes in the endoplasmic reticulum. Osmium postfixation may reduce or even abolish antigenicity in plastic-embedded tissue. After cryoultramicrotomy, however, even after osmium fixation, antibody may be used 1000 times more diluted than after plastic embedding. Embedding preserves ultrastructure and limited antigenicity while the use of cryoultramicrotomy is a far more sensitive technique.     

Growing and embedding monolayer cells in situ in beem capsule caps for light and electron microscopy

A method of growing and embedding monolayer cells in situ on the inner surface of the Beem capsule for light and electron microscopy is described. The results demonstrate that wandering cells in tissue and collected cells in suspension are readily grown on the Beem capsule and embedded in situ by a slight modification of the routine embedding procedure. The method seems to be particularly suited for studies of interactions between growing monolayer cells and various substances, infectious agents, or other types of cells added during the incubation period, where disruption by scraping and pelleting, or enzymatic reaction to remove the cells, would prevent such data from being gathered. The method is also suited for light and electron microscopic studies of minute tissue or organs, such as dissected mosquito salivary glands, whose embedding by the routine procedure is difficult.     

Quetol 651: Not just a low viscosity resin

Quetol 651, a low viscosity epoxy resin, is miscible with alcohols, acetone, and water. It is versatile and can be used as a single epoxide or mixed with other epoxides and anhydrides. The most important characteristic is that the addition of Quetol 651 to a formulation results in a lower viscosity embedding medium and allows for good detection of antigenic activity. Properly formulated and mixed resins containing Quetol 651 have excellent sectioning properties and good beam stability. The decrease in viscosity lends to lower specific gravity of the embedding medium and less interfering electron density between specimen elements resulting in better spatial resolution. New formulations and viscosity data are presented and compared to long used, embedding formulations and the extensive uses of Quetol 651 are reviewed. Microsc. Res. Tech. 79:50–57, 2016. © 2015 Wiley Periodicals, Inc.     

Surfactants as resin modifiers and their effect on sectioning

Ease of cutting thin sections with glass knives is markedly improved if the embedding resin contains a surfactant such as lecithin. With lecithin, it is possible to cut 50–100 thin sections from the same place on the knife edge even after facing off the block with 1–2-μm-thick sections. Image quality is similar to that of the unmodified resin if the resin formula is optimized. If not, some chatter or a “mottled” appearance of the tissue image may be present. Lecithin does not significantly affect sectioning with a diamond knife or the appearance of the section in the microscope. The increased ease of sectioning with glass presumably will be translated to diamond knives in the form of an increased useful life of the cutting edge.     

Preparing sections of skeletal muscle for transmission electron analytical microscopy (TEAM) of diffusible elements.

Comparative morphological examination and elemental analysis were carried out in structural compartments of sections of skeletal muscles. These had been prepared either by conventional plastic embedding technique or by various methods of cryo-ultramicrotomy. The analyses were performed in a Philips EM 301 with an Edax energy-dispersive X-ray spectrometer. Spectra obtained from sections of plastic-embedded muscle depended on the reagents used for fixation and staining and were absent if these were omitted. Brief fixation with glutaraldehyde resulted in gross ionic changes, and sectioning of frozen material with trough liquid led to extraction of elements. Sections cut from unfixed and frozen muscle without trough liquid showed numerous peaks. (Mg, P, S, Cl, K, Ca). In the superficial parts of the fibres of freeze-dried sections reproducible spectral differences were found between different structures. Thus, rapid freezing of unfixed tissue, dry cutting in the frozen state, and freeze-drying should be the procedure of choice if data on diffusible ions are desired.