A sandwich-embedding method for oriented sectioning

High‐quality sections are indispensable for many scientific studies. Most published methods are often time‐consuming or require special devices. We present an easy, quick and low‐cost method for oriented embedding of thin structures using glycol methacrylate resin and self‐constructed, reusable embedding tools made of overhead transparencies. This technique allows for more flexibility in orientation than other methods, enabling precise transverse, longitudinal and even oblique sectioning.     

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.     

Cryoimmobilization and three-dimensional visualization of C. elegans ultrastructure

Caenorhabditis elegans is one of the most important genetic systems used in current biological research. Increasingly, these genetics‐based research projects are including ultrastructural analyses in their attempts to understand the molecular basis for cell function. Here, we present and review state‐of‐the‐art methods for both ultrastructural analysis and immunogold localization in C. elegans. For the initial cryofixation, high‐pressure freezing is the method of choice, and in this article we describe two different strategies to prepare these nematode worms for rapid freezing. The first method takes advantage of transparent, porous cellulose capillary tubes to contain the worms, and the second packs the worms in E. coli and/or yeast paste prior to freezing. The latter method facilitates embedding of C. elegans in a thin layer of resin so individual worms can be staged, selected and precisely orientated for serial sectioning followed by immunolabelling or electron tomography.     

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 determination of thickness and surface relief in reembedded sections of an epoxy- and a melamine-resin containing ferritin as size standard

Chemical and physical data of two electron microscopic embedding media (the non-polar epoxy resin Epon 812 and the polar melamine resin Nanoplast FB 101) suggest that less kinetic energy must be applied for cutting a section from a Nanoplast block than from an Epon block of the same hardness and that, consequently, the cutting qualities of Nanoplast are better. To test this hypothesis, normal and extremely thin sections of Epon- and Nanoplast-embedded horse spleen ferritin micropellets were reembedded and resectioned for a determination of thickness and surface roughness. The ease with which extremely thin sections can be cut from the Nanoplast resin (8 nm versus 15 nm in Epon) and the smooth surface of these sections support the hypothesis that the cutting quality of an embedding material is determined primarily by its energy balance, i.e. by the kinetic energy which must be introduced for sectioning and the bonding energy which is released exothermically from a polymer while being sectioned.     

Effects of different epoxies on avoiding wrinkles in thin sections of botanical specimens

Fold or wrinkles appear frequently in thin sections cut from botanical material embedded in epoxy resin such as Epon 812 (Polysciences) or EM-bed-812 (Electron Microscopy Sciences). Small wrinkles may occur more or less perpendicular to the cutting direction and mostly across the cell wall, causing problems in the examination of the ultrastructure. We studied the occurrence of wrinkles and folds by methodological sectioning, with regard to block hardness, use of Formvar-support film, and chloroform vapor. Methodological sectioning or changing the block hardness did not reduce the occurrence of wrinkles in sections. Chloroform vapor improved the result as did also the use of uncoated grid. Wrinkles and folds could be avoided completely by embedding the specimens in LX-112 (Ladd) or ERL-4206 (Polysciences) low-viscosity epoxy resins, in which case Formvar-coated grids could be used, and chloroform vapor was not necessary.     

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.     

Technical advances in the sectioning of dental tissue and of on‐section cross‐linked collagen detection in mineralized teeth

Immunohistochemical detection of cross‐linked fibrillar collagens in mineralized tissues is much desired for exploring the mechanisms of biomineralization in health and disease. Mineralized teeth are impossible to section when embedded in conventional media, thus limiting on‐section characterization of matrix proteins by immunohistochemistry. We hypothesized that by using an especially formulated acrylic resin suitable for mineralized dental tissues, not only sectioning of teeth would be possible, but also our recently developed immunofluorescence labeling technique would be amenable to fully calcified tissues for characterization of dentinal fibrillar collagens, which remains elusive. The hypothesis was tested on fixed rodent teeth embedded in Technovit 9100 New®. It was possible to cut thin (1 μm) sections of mineralized teeth, and immunofluorescence characterization of cross‐linked type I fibrillar collagen was selected due to its abundance in dentine. Decalcified samples of teeth embedded in paraffin wax were also used to compare immunolabeling from either method using the same immunoreagents in equivalent concentrations. In the decalcified tissue sections, type I collagen labeling in the dentine along the tubules was “patchy” and the signal in the predentine was very weak. However, enhanced signal in mineralized samples with type I collagen was detected not only in the predentine but also at the limit between intertubular dentine, within the elements of the enamel organ and subgingival stroma. This report offers advances in sectioning mineralized dental tissues and allows the application of immunofluorescence not only for on‐section protein detection but importantly for detecting cross‐linked fibrous collagens in both soft and mineralized tissue sections. Microsc. Res. Tech. 73:741–745, 2010. © 2009 Wiley‐Liss, Inc.     

Demonstration of pseudorabies virus DNA in the mouse inner ear by an in situ nucleic acid hybridization technique in plastic embedded bony material

This investigation is concerned with the possibility of identifying viral DNA using the in situ DNA hybridization method in methylmethacrylate-embedded material. As an experimental model we chose viral labyrinthitis produced by intranasal infection of the mouse with pseudorabies virus. Fixation and embedding methods specially adapted to this procedure and bony histology preparation technique (specimens by grinding or micromilling) made it possible to identify viral DNA directly morphologically and virologically in the inner ear. Quantitative microphotometric analyses of trans-sagittal sections of the entire skull after in situ DNA hybridization are presented and discussed here as an explicit method of investigating the path of distribution of viral DNA in the brain and the inner ear.     

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.     

Direct observation of liquid crystals using cryo‐TEM: Specimen preparation and low‐dose imaging

Liquid crystals (LCs) represent a challenging group of materials for direct transmission electron microscopy (TEM) studies due to the complications in specimen preparation and the severe radiation damage. In this paper, we summarize a series of specimen preparation methods, including thin film and cryo‐sectioning approaches, as a comprehensive toolset enabling high‐resolution direct cryo‐TEM observation of a broad range of LCs. We also present comparative analysis using cryo‐TEM and replica freeze‐fracture TEM on both thermotropic and lyotropic LCs. In addition to the revisits of previous practices, some new concepts are introduced, e.g., suspended thermotropic LC thin films, combined high‐pressure freezing and cryo‐sectioning of lyotropic LCs, and the complementary applications of direct TEM and indirect replica TEM techniques. The significance of subnanometer resolution cryo‐TEM observation is demonstrated in a few important issues in LC studies, including providing direct evidences for the existence of nanoscale smectic domains in nematic bent‐core thermotropic LCs, comprehensive understanding of the twist‐bend nematic phase, and probing the packing of columnar aggregates in lyotropic chromonic LCs. Direct TEM observation opens ways to a variety of TEM techniques, suggesting that TEM (replica, cryo, and in situ techniques), in general, may be a promising part of the solution to the lack of effective structural probe at the molecular scale in LC studies. Microsc. Res. Tech. 77:754–772, 2014. © 2014 Wiley Periodicals, Inc.     

Extremely thin layer plastification for focused‐ion beam scanning electron microscopy: an improved method to study cell surfaces and organelles of cultured cells

Since the recent boost in the usage of electron microscopy in life‐science research, there is a great need for new methods. Recently minimal resin embedding methods have been successfully introduced in the sample preparation for focused‐ion beam scanning electron microscopy (FIB‐SEM). In these methods several possibilities are given to remove as much resin as possible from the surface of cultured cells or multicellular organisms. Here we introduce an alternative way in the minimal resin embedding method to remove excess of resin from two widely different cell types by the use of Mascotte filter paper. Our goal in correlative light and electron microscopic studies of immunogold‐labelled breast cancer SKBR3 cells was to visualise gold‐labelled HER2 plasma membrane proteins as well as the intracellular structures of flat and round cells. We found a significant difference (p < 0.001) in the number of gold particles of selected cells per 0.6 m² cell surface: on average a flat cell contained 2.46 ± 1.98 gold particles, and a round cell 5.66 ± 2.92 gold particles. Moreover, there was a clear difference in the subcellular organisation of these two cells. The round SKBR3 cell contained many organelles, such as mitochondria, Golgi and endoplasmic reticulum, when compared with flat SKBR3 cells. Our next goal was to visualise crosswall associated organelles, septal pore caps, of Rhizoctonia solani fungal cells by the combined use of a heavy metal staining and our extremely thin layer plastification (ETLP) method. At low magnifications this resulted into easily finding septa which appeared as bright crosswalls in the back‐scattered electron mode in the scanning electron microscope. Then, a septum was selected for FIB‐SEM. Cross‐sectioned views clearly revealed the perforate septal pore cap of R. solani next to other structures, such as mitochondria, endoplasmic reticulum, lipid bodies, dolipore septum, and the pore channel. As the ETLP method was applied on two widely different cell types, the use of the ETLP method will be beneficial to correlative studies of other cell model systems and multicellular organisms.     

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.     

Direct visualization of receptor arrays in frozen-hydrated sections and plunge-frozen specimens of E. coli engineered to overproduce the chemotaxis receptor Tsr

We have recently reported electron tomographic studies of sections obtained from chemically fixed E. coli cells overproducing the 60‐kDa chemotaxis receptor Tsr. Membrane extracts from these cells prepared in the presence of Tween‐80 display hexagonally close‐packed microcrystalline assemblies of Tsr, with a repeating unit large enough to accommodate six Tsr molecules arranged as trimers of receptor dimers. Here, we report the direct visualization of the Tsr receptor clusters in (i) vitrified cell suspensions of cells overproducing Tsr, prepared by rapid plunge‐freezing, and (ii) frozen‐hydrated sections obtained from cells frozen under high pressure. The frozen‐hydrated sections were generated by sectioning at ?150 °C using a diamond knife with a 25° knife angle, with nominal thicknesses ranging from 20 to 60 nm. There is excellent correspondence between the spatial arrangement of receptors in thin frozen‐hydrated sections and the arrangements found in negatively stained membrane extracts and plunge‐frozen cells, highlighting the potential of using frozen‐hydrated sections for the study of macromolecular assemblies within cells under near‐native conditions.     

Confocal laser scanning microscopic analysis of ectopic sublingual gland‐like tissue inside the hamster submandibular gland

Based on its histochemical properties, the secretory portion of the hamster submandibular gland has been classified as seromucous cells. The presence of endogenous peroxidase (PO) reaction was shown in the nuclear envelope, cisternae of endoplasmic reticulum and Golgi apparatus. The 3,3′‐diaminobenzidene, tetrahydrochloride (DAB) method revealed bipartite secretory granules containing a PO‐positive dense core surrounded by a less dense halo in these cells. In the present investigation, serous and mucous‐like cells were found in resin‐embedded semi‐thin sections of the DAB‐reacted hamster submandibular gland. These sections were already on glass slides for routine light microscopic observations, therefore electron microscopic analysis could be unrealizable. We then used reflectance‐mode confocal laser scanning microscopy to visualize additional sites of PO activity as detected in these sections. Using this approach, we found mucous cells with PO activity‐negative secretory granules and seromucous cells with PO activity‐positive spot‐like secretory granules of the regular sublingual gland most frequently adjacent to the serous cells with typical electron‐dense secretory granules. These cells clearly differ from the seromucous cells with bipartite secretory granules and the granular duct cells with typical electron‐dense secretory granules of the hamster submandibular gland. Additionally, secretory endpieces of the ectopic sublingual gland‐like tissue empty into the duct of the hamster submandibular gland lobule. Thus, our findings suggest that a mass of sublingual gland tissue extends into the hamster submandibular gland during its development, and PO may be synthesized and secreted into the same duct. Microsc. Res. Tech. 76:1284–1291, 2013. © 2013 Wiley Periodicals, Inc.     

Freeze-substitution and the preservation of diffusible ions

Freeze-substitution of biological material in pure acetone followed by low-temperature embedding in the Lowicryls K11M and HM23 yields stable preparations well suited for sectioning and subsequent morphological and microanalytical studies. Transmission electron microscopy of dry-cut sections shows that diffusible cellular thallium ions (Tl⁺) of Tl⁺-loaded muscle are localized at similar protein sites in freeze-substituted as in frozen-hydrated preparations. A comparison of X-ray micro-analytical data obtained from freeze-dried cryosections and sections of freeze-substituted normal (potassium-containing) muscle shows that K⁺ ion retention in the freeze-substituted sample is highly dependent on the freeze-substitution procedure used; so far, in the best case, about 67% of the cellular K⁺ is retained after freeze-substitution in pure acetone and low-temperature embedding. It is concluded that the retention of diffusible cellular ions is dependent on their interactions with cellular macromolecules during the preparative steps and that ion retention may be increased by further optimizing freeze-substitution and low-temperature embedding.     

Expression of the male reproduction‐related gene in spermatic ducts of the blue swimming crab,Portunus pelagicus,and transfer of modified protein to the sperm acrosome

Expression of a sex‐specific gene in Macrobrachium rosenbergii (Mr‐Mrr), encoding a male reproduction‐related (Mrr) protein, has been identified in the spermatic ducts (SDs) and postulated to be involved in sperm maturation processes. M. rosenbergii is the only decapod that the expression and fate of the Mrr protein has been studied. To determine that this protein was conserved in decapods, we firstly used cloning techniques to identify the Mrr gene in two crabs, Portunus pelagicus (Pp‐Mrr) and Scylla serrata (Ss‐Mrr). We then investigated expression of Pp‐Mrr by in situ hybridization, and immunolocalization, as well as phosphorylation and glycosylation modifications, and the fate of the protein in the male reproductive tract. Pp‐Mrr was shown to have 632 nucleotides, and a deduced protein of 110 amino acids, with an unmodified molecular weight of 11.79 kDa and a mature protein with molecular weight of 9.16 kDa. In situ hybridization showed that Pp‐Mrr is expressed in the epithelium of the proximal, middle, distal SDs, and ejaculatory ducts. In Western blotting, proteins of 10.9 and 17.2 kDa from SDs were all positive using anti‐Mrr, antiphosphoserine/threonine, and antiphosphotyrosine. PAS staining showed they were also glycosylated. Immunolocalization studies showed Pp‐Mrr in the SD epithelium, lumen, and on the acrosomes of spermatozoa. Immunofluorescence staining indicated the acrosome of spermatozoa contained the Mrr protein, which is phosphorylated with serine/threonine and tyrosine, and also glycosylated. The Mrr is likely to be involved in acrosomal activation during fertilization of eggs. Microsc. Res. Tech., 2013. © 2012 Wiley Periodicals, Inc.     

Low‐temperature glycol methacrylate resin embedding method: A protocol suitable for bone marrow immunohistochemistry,PCR, and fish analysis

Molecular analyses such as fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) are demanded to improve diagnostic accuracy in addition to immunohistopathology of bone marrow (BM) trephine specimens. Conventional BM embedding method needs decalcification, and its procedure may impair tissue morphology and DNA quality. Here, we report an undecalcified method by which glycol methacrylate resin is polymerized at low temperature (4°C). Using this method, BM enzyme activity and antigenic determinants are well preserved, and moreover, DNA extracted from plastic embedding sections is suitable for PCR amplification and sequencing, FISH analysis can be well done because of the DNA integrity of BM sections. If working with BM trephine specimen, our protocol offers the possibility to combine superior morphology with modern molecular analysis. Microsc. Res. Tech. 73:1067–1071, 2010. © 2010 Wiley‐Liss, Inc.     

Correlation of some mechanical properties of embedding resins with their behaviour in microtomy

The technology of ultramicrotomy is now well established, but the properties of the resin that determine the different forces needed to generate a section have been neglected, although this process could introduce artefacts in the thin sections. We have investigated the principal resin dependent factors involved in the sectioning process and determined the related mechanical properties. Tensile experiments have given the best correlation with the sectioning quality of the resin: the elastic (Young's) modulus value (depending on polymer structure or hardening mode), the presence of a short plastic flow for a controlled fracture and enough flexibility to minimize shearing, and internal cracks, appear to be the main characteristic parameters. The ultrathin section seems to be generated by a process close to cleavage, favoured by the relative hardness of the embedding media, while machining and “true” sectioning requires softer resins. Consequently, the rupture follows the path of least resistance in the specimen-resin composite, providing sections with a surface relief. Embedded biological material copolymerizes with polycondensed matrix (epoxy resins), and, by reducing the heterogeneity, gives smoother sections. Embedments hardened by radical polymerization provide a rougher relief, since almost no copolymerization occurs, offering to the microtome a heterogeneous block with two constituents of very different mechanical properties. The surface relief seems to be an important factor in labelling, staining, and imaging, and more attention has to be paid for some improvements of the quality of the information provided by electron microscopy.     

Reflectance in situ hybridization (RISH): detection,by confocal reflectance laser microscopy,of gold-labelled riboprobes in breast cancer cell lines and histological specimens

A method for reflectance in situ hybridization (RISH) is presented. The importance of the method is demonstrated by results obtained on cytological and histological breast cancer specimens. Scattering reflectance signals from 1-nm colloidal-gold particles after RNA/RNA in situ hybridization, using digoxigenin-labelled riboprobes, were detected by confocal scanning laser microscopy. The mRNA expression of two ras-related genes, rho B and rho C, was analysed in human histological breast cancer specimens and in human breast cancer cell lines. Horizontal (x, y) and vertical (z) optical sections after three-dimensional imaging were used for visualization. A marked heterogeneity (between individual cells and between specimens) was noted for the expression of the rho B gene, both in cytological and in histological samples. On the other hand, rho C was always expressed and showed no heterogeneity. This method allows the identification of several cellular constituents in an heterogeneous tissue structure, as demonstrated by the simultaneous detection of rho B (or rho C) by reflectance and of DNA, cytokeratin and/or vimentin by fluorescence.