Epoxy-Slide Embedment of Cytochemically Stained Tissues and Cultured Cells for Light and Electron Microscopy

A new method is described for embedding stained tissue sections, cells, cultured cells or organ cultures in a special polyethylene mold to form epoxy microscope slides (cost-a-slides). Cast-a-slides in which biological specimens are embedded may be examined by light microscopy and individual optimally stained cells or tissue areas selected for examination by various modes of electron microscopy or X-ray microanalysis. Cultured cells or organs can be grown, fixed, stained and embedded in epoxy in the same cast-a-slide mold. The cast-a-slides can be stored conveniently in the same manner as glass microscopy slides.     

Epoxy-slide embedment of cytochemically stained tissues and cultured cells for light and electron microscopy

A new method is described for embedding stained tissue sections, cells, cultured cells or organ cultures in a special polyethylene mold to form epoxy microscope slides (cast-a-slides). Cast-a-slides in which biological specimens are embedded may be examined by light microscopy and individual optimally stained cells or tissue areas selected for examination by various modes of electron microscopy or X-ray microanalysis. Cultured cells or organs can be grown, fixed, stained and embedded in epoxy in the same cast-a-slide mold. The cast-a-slides can be stored conveniently in the same manner as glass microscopy slides.     

Aqueous aldehyde (Faglu) methods for the fluorescence histochemical localization of catecholamines and for ultrastructural studies of central nervous tissue.

Aqueous solutions combining a high concentration of formaldehyde (4%) with low concentrations of glutaraldehyde (0.5--01%) have been used to simultaneously localize amines by the formation of fluorescent products and to fix central nervous tissue for electron microscopy. The fluorescence reaction is produced by the aldehyde mixture at room temperature and the fluorescence is stable when the tissue is maintained in aqueous solution. This means that nerve cell bodies and terminal fields which contain catecholamines can be located accurately in vibratome sections at the light microscope level and, after further processing, can be examined under the electron microscope. With 1% glutaraldehyde in the aldehyde mixture, ultrastructural details are well preserved; there is no significant distortion of any component of the tissue. If vibratome or cryostat sections are dried against glass slides, the intensity of the fluorescence reaction is enhanced and the sections can be permanently mounted.     

A method of processing tissue sections for staining with cu-promeronic blue and other dyes, using CEC techniques, for light and electron microscopy

The application to connective tissues etc. of new histochemical and ultrastructural methods involving consecutive treatment with dye-salt baths, specific enzymes, antibodies and decalcifying fluids, is best carried out on sections on glass slides. This ensures uniform access of reagents to substrates and allows the accurate monitoring of the results, as well as exploiting the strengths of the newer reagents (e.g. Cupromeronic Blue) in that they are highly coloured as well as electron dense. However, very high losses of connective tissue sections usually occur in the various solutions, to the extent that many important investigations are rendered almost impossible because of the high cost of the reagents. We describe a technique which we use routinely, that gives very high recovery of sections of "difficult" tissues from multi-stage enzyme and stain treatments, for light microscopy followed by plastic embedding of the coloured sections for electron microscopy.     

Aqueous aldehyde (Faglu) methods for the fluorescence histochemical localization of catecholamines and for ultrastructural studies of central nervous tissue

Summary Aqueous solutions combining a high concentration of formaldehyde (4%) with low concentrations of glutaraldehyde (0.5–1%) have been used to simultaneously localize amines by the formation of fluorescent products and to fix central nervous tissue for electron microscopy. The fluorescence reaction is produced by the aldehyde mixture at room temperature and the fluorescence is stable when the tissue is maintained in aqueous solution. This means that nerve cell bodies and terminal fields which contain catecholamines can be located accurately in vibratome sections at the light microscope level and, after further processing, can be examined under the electron microscope. With 1% glutaraldehyde in the aldehyde mixture, ultrastructural details are well preserved; there is no significant distortion of any component of the tissue. If vibratome or cryostat sections are dried against glass slides, the intensity of the fluorescence reaction is enhanced and the sections can be permanently mounted.     

A Simple Technique for Osmicating and Flat Embedding Large Tissue Sections for Light and Electron Microscopy

Many investigators now use thin hand-sliced, tissue chopper, or Vibratome sections of fresh tissue in various procedures. In our experience brain and nerve sections varying in thickness from less than 40 to more than 300 μm, with or without prior embedding in agar, have a tendency to roll up or curl during aldehyde fixation and buffer washes. Once osmicated, such curled sections cannot be flattened. When the entire cut face of such thin slices is to be studied, sufficiently flat embedding so that some regions are not completely sectioned before others are even sampled is critical. This report describes fixation and flat embedding procedures, developed for light and electron microscopic autoradiographic studies of plastic embedded brain slices about 200 μm thick (Schwartz 1981), which can be applied to any comparable thin slice of nervous tissue (or potentially of many other tissues) to achieve maximally flat tissue faces. Since osmicated tissue slices are usually too thick to be transilluminated for direct examination with the light microscope, the methods described simplify preparation of the semithin sections required for this purpose.     

Superficial Warming of Epoxy Blocks for Cutting of 25-150 μm Sections to be Resectioned in the 40-90 nm Range

An improved method is described in which tissue areas can be initially identified in thick sections by light microscopy and isolated for subsequent ultrathin sections and observation by electron microscopy. This is achieved by embedding in hard Epon which can be sectioned at 25-150 μm on a sliding microtome after softening the blockface by applying a 60-70 C tacking iron to its surface immediately before each section is taken. The thick sections are then mounted on plastic slides to enable light microscopic selection of areas to be observed by electron microscopy. The selected areas are remounted on faced Epon blanks and resectioned at less than 50 nm. This technique makes it possible to obtain thick sections while maintaining an Epon hard enough for good serial ultrathin sections.     

A Method for Processing Light Microscopy Sections for Oriented Ultramicrotomy

A simple technique is described for processing optical microscopy sections attached to glass slides for ultramicrotomy in any desired plane. A silicone rubber mold with a central orifice is clamped onto the slide so that the orifice overlies the section. Routine processing and embedding procedures are carried out in the well formed by the orifice.     

Preparation of methacrylate-embedded cytologic smears from prefixed cells

A new method of preparing smears of alcohol-fixed cytologic material by using methacrylate embedding medium to make the cells adhere on plain glass slides is presented. After centrifugation, the cytologic material was mixed with Lowicryl K4M embedding medium and smeared on slides. The polymerization process was achieved by exposing the slides to ultraviolet light. The morphology in such smears was similar to that of specimens prepared by the filter technique. The methacrylate method does not have the most common disadvantages of the filter technique--the development of air bubbles over time and the visually disturbing presence of the filter.     

Ultrastructural localization of neurophysin-like immunoreactivity in the rat posterior pituitary. An alternative method using frozen-dried tissue fixed with OsO4 vapor.

Electron microscopic identification of elements containing neurophysin-like immunoreactivity can be accomplished in rat posterior pituitary that has been frozen-dried and fixed with OsO4 vapor. Alternating serial ultrathin sections are placed on grids and glass slides. The sections on the slides are stained for neurophysin using immunofluorescence histochemistry, and the resultant images are superimposed on electron micrographs from the adjacent sections. The method provides several advantages for the localization of neuropeptide immunoreactivity in nervous tissue.     

刺螫库蠓成虫内部器官系统组织学观察

【目的】刺螫库蠓Culicoides punctatus是一种重要媒介蠓虫,是施马伦贝格病毒(Schmallenberg virus, SBV)的主要传播载体。通过石蜡切片和苏木素 伊红染色技术观察刺螫库蠓成虫内部器官系统的组织结构。【方法】利用网扫法、灯诱法采集刺螫库蠓成虫,除足和翅外置于Duboscq-Brasil固定液中常温固定,然后经过逐级脱水、二甲苯透明、石蜡包埋、连续切片以及苏木素-伊红染色等过程制成玻片,利用光学显微镜进行观察和拍照。【结果】刺螫库蠓成虫消化系统不存在性别差异,由消化道及唾液腺组成。中枢神经系统由脑和腹神经索组成。脑可划分为前脑、中脑和后脑3个功能区。复眼和触角是刺螫库蠓主要的感觉器官。腹神经索可分为咽下神经节、胸神经节和腹神经节。呼吸系统主要由气管组成,气管遍布全身,无肺组织,胸部具有2对气门,分别位于中胸和后胸;腹支囊组织呈泡状,着色不明显。生殖系统包括内生殖器官和外生殖器官,其中雌性内生殖器官包括卵巢、输卵管、受精囊及生殖附腺,雄性内生殖器官包括精巢、输精管、射精管及生殖附腺。【结论】本研究明确了刺螫库蠓成虫的消化系统、神经系统、呼吸系统、生殖系统以及感觉器官的结构特征,为库蠓的发育研究提供了更为直接、准确的证据,有助于提高蠓媒监测、预报及防制的全面性和精确性。     

A rapid immunogold-silver staining for detection of bromodeoxyuridine in large numbers of plastic sections, using microwave irradiation

Summary A rapid and convenient method for the large scale, immunogold-silver staining (IGSS) of bromodeoxyuridine (BrdU) incorporated by S phase cells, by means of a monoclonal antibody (anti-BrdU) is described. Nineteen slides at a time can be incubated with the antibodies and the protein A-gold (PAG) in staining jars. The antibody and protein A-gold solutions could be used at least five times to incubate new batches of slides. The incubation times with these solutions were shortened by means of microwave irradiation. In this way 200 slides carrying at least 800 sections could be easily processed under the same conditions in one day, using 1.25ml neat antibody solutions of anti-BrdU and rabbit anti-mouse.For light microscopy bothpplastic embedding systems: methylmethacrylate (MMA) and glycolmethacrylate (GMA) can be stained with this technique. The MMA sections, of which the plastic has to be removed before the IGSS, has the advantage of a stronger labelling intensity. The GMA plastic, which contains a cross-linking, agent cannot be removed and consequently for GMA sections it is necessary to incubate the sections with a proteolytic enzyme (trypsin) before the IGSS, to reexpose the antigenic binding sides. However, the GMA sections can be allowed to air dry during the IGSS without negative effects on the morphology. This makes it possible to perform the antibody and the PAG-incubating steps on one day and to finish the IGSS the next day. In this way twice as many GMA slides can be incubated with the same antibody and PAG solutions than with MMA slides.In both plastic embedding systems the intensity of the BrdU labelling was found to be stronger in Carnoy's than in Bouin's fixed sections.     

A perfusion-fixation procedure for the concurrent demonstration of Timm's, horseradish peroxidase (HRP), and acethycholinesterase (AChE) histochemistry

The sulfide-silver method of Timm has been a widely used histochemical technique to demonstrate the presence of heavy metals in biological tissue, particularly in the central nervous system. However, the use of this method or its several modifications results in less than optimal morphological preservation and requires embedding the tissue in paraffin or freezing it and cutting it directly onto slides with a cryostat. These procedures can decrease the sensitivity and limit the application of other histochemical procedures, particularly when experiments necessitate processing large specimens or reaction procedures require techniques using free-floating sections. A perfusion-fixation protocol is described that yields sufficient fixation to cut whole frozen blocks of tissue with a sliding microtome, permits the use of free-floating sections, and allows the concurrent demonstration of horseradish peroxidase and acetylcholinesterase histochemistry without loss of sensitivity. The method consists of a short initial exposure to a sodium sulfide solution followed by a prolonged exposure to a combined sulfide-aldehyde fixative solution.     

Protocol for the combination of neurohistological techniques on vibratome obtained sections

Introduction. The histological study of the nervous system requires the use of special techniques. Currently, no methods are available to visualize simultaneously all the cellular constituents of nervous tissue. Objectives. A protocol was adapted for staining nervous tissue by modification of a formerly difficult procedure. Materials and methods. Slices of brain and spinal cord, 4 mm thick, were taken from adult mice, previously fixed by intracardiac perfusion with 4% paraformaldehyde. Vibratome sections were obtained with thickness of 15-25 μm. These were mounted on glass slides prepared with gelatin as an adhesive. The preparations were subjected to staining protocol Luxol Fast Blue-PAS-hematoxylin (LPH) combined with silver staining method (LPH-Holmes). Results. LPH technique yielded an excellent differentiation of gray and white matter in all regions of the nervous system. A panoramic view of the gray matter was colored pink in contrast to the myelinated nerve fibers and tracts which were light blue. The combination LPH-Holmes retained the staining characteristics but significantly improved the demarcation of axons and tracts. Conclusions. A protocol was standardized for the LPH and LPH-Holmes nervous tissue stains applied in combination to tissue slices obtained with a vibratome. The method was shorter, less wasteful and less expensive than the original and also better preserved the integrity of nervous tissue.     

Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits

Many biological functions depend critically upon fine details of tissue molecular architecture that have resisted exploration by existing imaging techniques. This is particularly true for nervous system tissues, where information processing function depends on intricate circuit and synaptic architectures. Here, we describe a new imaging method, called array tomography, which combines and extends superlative features of modern optical fluorescence and electron microscopy methods. Based on methods for constructing and repeatedly staining and imaging ordered arrays of ultrathin (50-200 nm), resin-embedded serial sections on glass microscope slides, array tomography allows for quantitative, high-resolution, large-field volumetric imaging of large numbers of antigens, fluorescent proteins, and ultrastructure in individual tissue specimens. Compared to confocal microscopy, array tomography offers the advantage of better spatial resolution, in particular along the z axis, as well as depth-independent immunofluorescent staining. The application of array tomography can reveal important but previously unseen features of brain molecular architecture.     

Microscopic investigations of the interaction of proteins with surfaces

The application of light microscopy to the study of protein-surface interactions is described with several examples showing its versatility. Extensive use is made of image analysis algorithms to extract quantitative information from the digital images and it is shown how this can shed light on the processes taking place on the surface. Amongst the surfaces investigated are siloxane films on silicon wafers, glucose oxidase/poly(ethyleneglycol) films cast onto glass slides and glucose oxidase entrapped in electropolymerised poly(phenyleneoxide) films on platinum. Parameters that can be measured include: surface loading, surface heterogeneity, distribution of enzyme activity, surface mobility and film porosity. Both reflected light and fluorescence microscopy were used to characterise the surfaces and it was concluded that not only does light microscopy offer a powerful tool for the characterisation of surfaces but it may also provide an interface between bioelectronic materials and conventional computing machinery.     

A novel thin section preparation and staining protocol to increase contrast and resolution of cell details for light microscopy

ABSTRACT

We developed a novel sectioning and staining method to make high contrast, high resolution sections of plant tissue for light microscopy. Specimens of teosinte (Zea mays L., ssp. mexicana) root tips were fixed and embedded in Technovit 7100? plastic resin. Thin sections, 1?2.5 μm, were cut and mounted on glass slides. The sections were either treated with RNase or not, then stained with 0.1% toluidine blue O and observed through ∞/0 objective lenses. For light microscopy, the enzyme staining procedure increased resolution and contrast. High magnification ∞/0 objective lenses produced high quality images for digital photography without using a coverslip or immersion oil. Our slide preparation and microscopic analysis were less labor intensive and more rapid than previous methods and enabled rapid and precise alignment of serial transverse sections for both tracking cell lineages and tissue measurements.     

A Quick Embedding Method for Light and Electron Microscopy of Textile Fibers

A quick embedding method employing UV polymerization reactions has been devised for embedding fibers in acrylic and meth-acrylate media. The resultant thin, flat embed-dings are suitable for both light and electron microscopy.     

A Quick Embedding Method for Light and Electron Microscopy of Textile Fibers

A quick embedding method employing UV polymerization reactions has been devised for embedding fibers in acrylic and meth-acrylate media. The resultant thin, flat embed-dings are suitable for both light and electron microscopy.     

A Golgi-Electron Microscope Method for Insect Nervous Tissue

Golgi's light microscopic method of selective silver impregnation for nervous tissue combined with electron microscopy appears to offer a promising method for working out the detailed anatomy of individual neurons and their connections. Insect nervous tissue is fixed in a mixture of 2% paraformaldehyde and 21/2% glutaraldehyde in Millonig's buffer (pH 7.2) before postfixation for 12 hours in a solution brought to pH 7.2 with KOH containing 2% potassium dichromate, 1% osmium tetroxide and 2% D-glucose. The tissue is then transferred to a solution of 4% potassium dichromate for 1 day; and for 1-2 days to a 0.75% silver nitrate solution. After dehydration and embedding in Araldite, 50μm sections am made. Areas of interest are cut from these sections and re-embedded in silicone molds. Ultrathin sections are then cut and stained with uranyl acetate and lead citrate. The Golgi method described here gives good results at the level of both light and electron microscopy.