Lectins for histochemical demonstration of glycans

Lectins have been proven to be invaluable reagents for the histochemical detection of glycans in cells and tissues by light and electron microscopy. This technical review deals with the conditions of tissue fixation and embedding for lectin labeling, as well as various markers and related labeling techniques. Furthermore, protocols for lectin labeling of sections from paraffin and resin-embedded tissues are detailed together with various controls to demonstrate the specificity of the labeling by lectins.     

Polychromatic staining of epoxy semithin sections: a new and simple method

A simple, rapid method is described for the polychromatic coloration of semithin sections, which is applicable to material routinely processed for transmission electron microscopy. Material fixed with a glutaraldehyde-paraformaldehyde mixture and postfixed in osmium tetroxide with or without potassium ferrocyanide and embedded in different types of resin (Durkupan-ACM, Spurr resin, Taab resin) can be used. Constant and homogenous results are obtained with this technique, the staining procedure being achieved at room temperature in no more than 10 min. Sections of 0.5–1 m in thickness are oxidised and bleached. After washing, sections are stained in two steps with carbol methylene blue/carbol gentian violet solution and pararosaniline solution. Using the method described in this paper, a polychromatic coloration of the different cells and tissues was obtained (epithelial cells in various shades of blue-violet, connective tissue and elastic laminae of blood vessels in pink or red, etc.). This procedure provides greater contrast between cytoplasm and nuclei, and among the different types of cells and tissues than is seen with toluidine blue, which is very useful for observation and photography of semithin sections. Polychromatic methods found in the literature are normally complex and require a lengthy staining time or cannot be applied on material routinely processed for transmission electron microscopy. Our method is simple, rapid and can be used on any type of material routinely processed for transmission electron microscopy and embedded in epoxy resins.     

The cryomicrotomy of rat dental tissues: a technique for histological and immunohistochemical analysis

Summary We have applied a method developed for the cryomicrotomy of non-decalcified bone to the histological preparation of the tooth and related dental tissues. Cryosections of rat mandible have been cut on a heavy-duty freezing microtome. Both cellular and extracellular structure was well preserved and the sections of tooth and bone appeared to be suitable for optical and scanning electron microscopy and for immunohistochemical analysis. However, there was an overall strong non-specific binding of immunohistochemical reagents to enamel which was not evident in the other mineralized tissues of the mandible. This may relate to important differences in the nature of this tissue.     

Fixation and tissue preservation for antibody studies

Synopsis The methods of fixation and preparation of lymphoid tissues for the immuno-enzyme technique are reviewed. For this technique an enzyme is used first as an antigen and then as a marker to demonstrate its specific antibody. A variety of commonly employed fixatives satisfactorily conserve tissues for the light microscopic detection of antibody but, for electron microscopy, glutaraldehyde or formaldehyde or both are the fixatives of choice. The main technical problem for electron microscopy is to reduce the size of the tissue fragments sufficiently so that the enzymes and their substrates permeate through the fixed tissues. The merits and short-comings of the different preparative techniques are examined and it is shown that the most reproducible results are obtained with 40 m frozen sections. Some of the problems of non-specific staining arising from fixation procedures, as well as endogenous enzyme activity, are discussed. The evidence for and against antibody inactivation by fixation and enzyme inactivation by interaction with its specific antibody is reviewed.     

Technique for Revealing the Three-Dimensional Architecture of Whole Preserved Spiculated Invertebrates

Procedures for revealing the three-dimensional arrangement of calcareous sclerites, spicules, or ossicles embedded within connective tissue in formalin fixed invertebrates are described. Spicules are stained with alizarin red S following maceration of preserved animals or colonies with either trypsin or KOH solutions. Connective tissue is stained with alcian blue in different samples prior to maceration. Stained animals or colonies are cleared in glycerin. This method for revealing spicular structure and arrangement and the gross morphology of connective tissues offers several advantages over either scanning electron microscopy or reconstruction from serial sections.     

Morphological and immunological confirmation of the presence of chlamydiae in the gut tissues of the chesapeake bay clam,Mercenaria mercenaria

The original electron microscopic identification by other investigators in 1977 of chlamydiae in the gut tissues of the Chesapeake Bay hard clam (Mercenaria mercenaria) is corroborated and further supported by evidence ofChlamydia-specific immunofluorescence (IF). Our electron microscopy demonstrated that gut tissue cells were heavily infected with chlamydiae in all stages of development but the intrachlamydial phage-like particles reported in 1977 were not seen. Tissue sections stained with IF reagents were strongly positive, and IF was blocked in varying degrees with chlamydial antisera produced in a goat and a turkey. The IF-positive tissue sections contained intracytoplasmic inclusions that stained darkly with Lugol's iodine (indicating the presence of glycogen) while IF-negative tissues had little if any iodinestaining material. Furthermore, electron micrographs of chlamydiae-containing phagosomes showed numerous rosettes of electron-dense particles typical of glycogen. The presence of iodine-positive phagosomes with electron dense rosettes suggests that the organisms are glycogen-producing chlamydiae biochemically related toChlamydia trachomatis. Repeated attempts to cultivate chlamydiae from the clam tissues in cell cultures and laboratory animals failed.     

The microfibrils of connective tissue: II. Immunohistochemical detection of the amyloid P component

Immunohistochemical methods were used for the detection of the amyloid P component in the microfibrils of two regions: the zonule of the eye and the connective tissue of the foot pad in 20- to 50-gm mice. Following fixation by immersion in 4% formaldehyde, the eyes and foot pads were embedded in paraffin, and sections were immunostained for light microscopy by using antiamyloid P component antiserum followed by peroxidase-antiperoxidase procedure. For electron microscopy, formaldehyde-fixed tissues were immunostained for the amyloid P component with protein A-gold by using either thin Lowicryl sections or frozen sections which were then embedded in Epon for thin sectioning. In the zonule of the eye, the light microscope showed that zonular fibers were strongly immunostained for the amyloid P component; there was also weak staining of the nonpigmented ciliary epithelium at the distal end of the fibers and of the zonular lamella at their proximal end. The electron microscope revealed clear-cut immunolabeling of the microfibrils making up zonular fibers as well as of individual microfibrils. In the foot pad, the light microscope detected a weak diffuse staining of connective tissue, whereas the electron microscope showed immunolabeling restricted to microfibrils. It was concluded that the amyloid P component was present in, or associated with, microfibrils. Purified amyloid P component was prepared and examined in the electron microscope after either negative staining or routine processing. After negative staining, it appeared as flat pentagonal units, frequently associated into columns. After routine processing, the units looked like cross sections of microfibrillar tubules. The dimensions of the units matched those of the hypothetical segments of the tubules. It was concluded that this tubule consisted of a column of amyloid P units. The cohesion of the units within the column was likely to be reinforced by the bands present at the surface of microfibrils.     

Bovine olfactory and nasal respiratory epithelium surfaces

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

Polystyrene embedding: a new method for light and electron microscopy.

Polystyrene embedments of histological specimens can be obtained with a solution of 1:14 polystyrene-toluene, 5% benzyl alcohol and 1% dibutyl phthalate, allowing the solvent to evaporate in polyethylene containers for 2-3 days at 58 C. The resulting blocks are easily cut into truncated pyramids, each containing a piece of tissue, which are then glued to a Plexiglas support. Drying is completed at 80 C for 20 hr. The pyramids can then be sectioned to produce thick sections with a steel knife or to produce semi- or ultrathin sections with a glass knife. A 10% paraldehyde solution is used to mount the light microscopy sections on a slide heated on a hot plate to 80 C; these can be treated with the same techniques used with paraffin sections. The results are of high quality. Semithin sections of tissues fixed for electron microscopy can be stained directly after mounting, or by a wider range of stains once the polystyrene has been removed by organic solvents. In electron microscopy, the ultrathin sections obtained with the usual techniques are highly electron beam-resistant and given acceptable results.     

Review of problems of correlations of structure and function in cells

Many of the correlations in cytology between structure and function compare the metabolism of subcellular fractions with the electron microscopy of the same tissues. Conclusions from subcellular fractionation have generally been derived on the assumption that killing the animal or plant, homogenisation, centrifugation and extraction of the tissues, do not affect the activities of the biochemical structures examined nor their distribution in the subcellular fractions; this assumption has never been tested. There is insufficient literature on the effects of reagents used in histology, immunocytochemistry and electron microscopy, on chemical activities, their distribution, or their visibility in sections. The artifacts of electron microscopy, such as shrinkage and precipitation, are widely recognised but not taken into account sufficiently when structures are examined. No attempts have been made to explain the two dimensional geometry of many 'unit' membranes, nor how a cytoskeleton can cause intracellular movements. Particular examples, such as the alleged location of oxidative phosphorylation along the mitochondrial cristae, the presence of biochemical ribosomal and lysosomal activities without the appearance of ribosomes and lysosomes, and intracellular movements in the believed presence of a cytoskeleton, are given to illustrate the difficulties of widely believed correlations between structure and function in cells.     

Combined light and electron microscope immunocytochemical localization of scattered peptidergic neurons in the central nervous system

A pre-embedding immunocytochemical technique is described for combined light and electron microscope study of peptidergic neurons in the central nervous system. The protocol is especially designed to overcome the sampling problems inherent in electron microscope study of structures, such as luteinizing hormone-releasing hormone (LHRH) neurons, that are scattered individually across large brain regions. The fixation methods outlined for several mammalian species include immersion and vascular perfusion with acrolein. Fine-structural preservation and LHRH immunoreactivity obtained with this fixative are compared to results with more conventional fixatives. Vibratome sectioning and a "pretreatment" regime, which prepare the tissues for immunocytochemistry, are described. Immunocytochemical labeling is done with free-floating sections and the peroxidase-antiperoxidase unlabeled antibody enzyme technique. Techniques are also described for the subsequent processing of immunoreacted sections for electron microscopy. These methods ensure that the processed sections are readily scanned by light microscopy, so that regions containing immunoreactive structures can be specifically chosen for electron microscope analysis. Sample electron micrographs are shown that illustrate some fine structural features of LHRH neurons in rats, bats, ferrets, and monkeys, as revealed with the techniques described.     

A Study of Criteria Permitting the Use of Plastinated Specimens for Light and Electron Microscopy

Plastination permits the preservation of anatomical specimens in a physical state approaching that of the living condition. We studied the possibility of using silicone plastinated fragments of spleen and pancreas for optical and electron microscopy, and found that with an adequate fixation protocol, plastinated specimens can be used for both light microscopy and ultra-structural studies. Deplastination with sodium methoxide permitted production of clean sections. Artifacts produced by plastination/deplastination could be nearly eliminated by glutaraldehyde/formaldehyde fixation. The (Biodur) silicone S10 polymer is transparent and stable in an electron beam, and plastinated tissues can be contrasted or colored similar to tissues embedded in Epon 812. In addition to being very life-like, plastinated tissues are stable and easy to handle. They can also be used for electron and light microscopic studies. This technique may also allow retrospective epidemiological studies of archived pathology specimens.     

Fluorescence photooxidation with eosin: a method for high resolution immunolocalization and in situ hybridization detection for light and electron microscopy

A simple method is described for high-resolution light and electron microscopic immunolocalization of proteins in cells and tissues by immunofluorescence and subsequent photooxidation of diaminobenzidine tetrahydrochloride into an insoluble osmiophilic polymer. By using eosin as the fluorescent marker, a substantial improvement in sensitivity is achieved in the photooxidation process over other conventional fluorescent compounds. The technique allows for precise correlative immunolocalization studies on the same sample using fluorescence, transmitted light and electron microscopy. Furthermore, because eosin is smaller in size than other conventional markers, this method results in improved penetration of labeling reagents compared to gold or enzyme based procedures. The improved penetration allows for three-dimensional immunolocalization using high voltage electron microscopy. Fluorescence photooxidation can also be used for high resolution light and electron microscopic localization of specific nucleic acid sequences by in situ hybridization utilizing biotinylated probes followed by an eosin-streptavidin conjugate.     

Periostin regulates collagen fibrillogenesis and the biomechanical properties of connective tissues

Periostin is predominantly expressed in collagen-rich fibrous connective tissues that are subjected to constant mechanical stresses including: heart valves, tendons, perichondrium, cornea, and the periodontal ligament (PDL). Based on these data we hypothesize that periostin can regulate collagen I fibrillogenesis and thereby affect the biomechanical properties of connective tissues. Immunoprecipitation and immunogold transmission electron microscopy experiments demonstrate that periostin is capable of directly interacting with collagen I. To analyze the potential role of periostin in collagen I fibrillogenesis, gene targeted mice were generated. Transmission electron microscopy and morphometric analyses demonstrated reduced collagen fibril diameters in skin dermis of periostin knockout mice, an indication of aberrant collagen I fibrillogenesis. In addition, differential scanning calorimetry (DSC) demonstrated a lower collagen denaturing temperature in periostin knockout mice, reflecting a reduced level of collagen cross-linking. Functional biomechanical properties of periostin null skin specimens and atrioventricular (AV) valve explant experiments provided direct evidence of the role that periostin plays in regulating the viscoelastic properties of connective tissues. Collectively, these data demonstrate for the first time that periostin can regulate collagen I fibrillogenesis and thereby serves as an important mediator of the biomechanical properties of fibrous connective tissues.     

Selection of a simple protease procedure for identifying mast cells in routinely processed human tissues

Proteases present in mast cell granules have been harnessed to demonstrate mast cells in human tissues. A number of substrate mixtures were tested. D-Val-Leu-Arg-4-methoxy-2-naphthylamide (MNA) plus Fast Blue B was the best for identifying human mast cells, yielding the most specific and complete staining. The procedure is simple and the results are permanent. Cryostat sections of aldehyde-fixed routine preparations or paraffin sections of Carnoy-fixed tissues give the most satisfactory results. Mast cells are stained a strong red color that stands out distinctly from the surrounding tissues, so that they can be easily identified by simple microscopy. A double-staining technique, first for protease and subsequently using Alcian Blue, showed that as progressive protease staining occurs, the alcianophilia of mast cells is lost. This procedure demonstrated that mast cells in the mucosa of human gut generally required longer incubations to develop protease staining than in other connective tissue sites. In post-mortem tissues, mast cells retain their protease activity well and so can be demonstrated in cryostat sections of aldehyde-fixed material, giving a more complete picture than with Alcian Blue. The synthetic substrate D-Val-Leu-Arg-MNA can be recommended for routine identification of mast cells in human tissues.     

Lectin--digoxigenin conjugates: a new hapten system for glycoconjugate cytochemistry.

We report the use of a novel hapten system for lectin cytochemistry. Various lectins conjugated to the steroid hapten digoxigenin (DIG) and monospecific anti-digoxigenin antibodies were applied for the light and electron microscopic detection of glycoconjugates in tissue sections. Both IgG and Fab' anti-DIG antibodies were complexed to particles of colloidal gold and compared to commercially available alkaline phosphatase and horseradish peroxidase conjugated Fab' as general second step reagents. The three different markers performed equally well on paraffin sections whereas the gold-labeled antibodies were superior reagents for semithin and ultrathin sections of Lowicryl K4M embedded tissues. In conjunction with the latter marker, no pretreatment to abolish endogenous enzyme activity was necessary. At the light microscope level, gold signal amplification by the photochemical silver reaction was required. DIG, in contrast to biotin, does not occur in animal tissues thus eliminating the need for blocking reactions prior to lectin incubation. Compared to affinity techniques using glycoprotein-gold complexes as second step reagent the DIG hapten system required smaller amounts of lectins. The staining patterns were indistinguishable from those obtained in other lectin-gold techniques and the specificity of the labeling could be demonstrated in sugar inhibition tests.     

Assessment of the Acrylic Resin Technovit 7200 VLC for Studying the Gingival Mucosa by Light and Electron Microscopy

Technovit 7200 VLC is an acrylic resin formulated for embedding undecalcified hard tissues which are prepared for light microscopy according to a cutting-grinding technique. To employ this resin for embedding and cutting soft tissues by ultramicrotomy, we carried out a qualitative study on biopsies of canine gingival mucosa using light and transmission electron microscopy. For a critical evaluation of this resin, some biopsies were embedded in Agar 100, an epoxy resin widely used in morphological studies. At the light microscopic level the samples embedded in Technovit 7200 VLC showed good morphology and excellent toluidine blue staining of different cell types and extracellular matrix. At the ultrastrueturallevel, nuclei, cytoplasmic organelles, collagen fibrils and ground substance appeared well preserved and showed high electron density. The acrylic resin was stable under the electron beam and its degree of shrinkage appeared to be very low. We conclude that Technovit 7200 VLC can be employed for ultramicrotomy for both light and electron microscopic investigation of soft tissues.     

Assessment of the Acrylic Resin Technovit 7200 VLC for Studying the Gingival Mucosa by Light and Electron Microscopy

Technovit 7200 VLC is an acrylic resin formulated for embedding undecalcified hard tissues which are prepared for light microscopy according to a cutting-grinding technique. To employ this resin for embedding and cutting soft tissues by ultramicrotomy, we carried out a qualitative study on biopsies of canine gingival mucosa using light and transmission electron microscopy. For a critical evaluation of this resin, some biopsies were embedded in Agar 100, an epoxy resin widely used in morphological studies. At the light microscopic level the samples embedded in Technovit 7200 VLC showed good morphology and excellent toluidine blue staining of different cell types and extracellular matrix. At the ultrastrueturallevel, nuclei, cytoplasmic organelles, collagen fibrils and ground substance appeared well preserved and showed high electron density. The acrylic resin was stable under the electron beam and its degree of shrinkage appeared to be very low. We conclude that Technovit 7200 VLC can be employed for ultramicrotomy for both light and electron microscopic investigation of soft tissues.     

Unconventional application of standard light and electron immunocytochemical analysis to aldehyde-fixed, araldite-embedded tissues

A simple procedure for the immunocytochemical analysis of glutaraldehyde/formaldehyde-fixed, Araldite- or Epon-embedded tissues by either light or electron microscopy is presented. Retention of immunoreactive antigen in deplasticized sections was achieved by use of a low concentration of glutaraldehyde in the fixative in combination with a seldom-used plastic solvent. This protocol produced good ultrastructural preservation in tissues and large, high-quality, 2-micrometers thick, plastic-free sections. These semithin sections provided a level of structural and antigenic preservation, image resolution, and labeling intensity that surpassed all other conventional sectioning methods used for immunocytochemistry. The capacity to use a single tissue sample in studies designed for light and electron immunocytochemistry, in conjunction with existing autoradiographic and cytochemical techniques, makes this a very desirable method for routine tissue preparation in research and clinical applications.     

IMPROVED TECHNIQUES FOR THE PREPARATION OF ULTRATHIN FROZEN SECTIONS

Ultrathin frozen sections of biological tissues for electron microscopy provide certain advantages in cytochemical studies in which the penetration of cells by large molecules is necessary and in morphological studies of cellular constituents which are dissolved by the reagents employed in routine plastic embedding. The recent introduction of several types of commercially available cryo-ultramicrotomes makes it possible for many laboratories to employ this valuable tool. This paper summarizes recent improvements in the methods developed in this laboratory for preparing ultrathin frozen sections and reviews some of the inherent problems involved in their use. These procedures may serve as a baseline for other investigators who can then modify or adapt them for their specific purposes.