Notes on Technic: Simple, Reliable Detection of Latex Microspheres in High Quality Tissue Sections

Latex microspheres used in biological research have been visualized by light microscopy in mounts of cell suspensions, disrupted cells, or cleared tissues (Mishima et al 1987, Koonce et al 1986, LeFevre et al 1978); in unembedded coverslip monolayers (Koerten et al 1980); in fixed (Cornwall and Phillipson 1988) or unfixed (Wells et al 1988) frozen sections; in paraffin sections cleared and deparaffinized with n-butyl alcohol (Callebaut and Meeussen 1989); and in tissues embedded in resins suitable for transmission electron microscopy, such as Spurr's (Hampton et al 1987), Epon (Herzog and Miller 1979), or Ladd Low Viscosity Epon (LeFevre et al 1985). Paraffin embedding, and some plastic embedments, are impractical for demonstration of latex beads because the beads are dissolved by such organic solvents as xylene, dioxane, or chloroform (Van Furth and Diesselhoff-Den Dulk 1980), propylene oxide (Lentzen et al 1984), amyl acetate (Okada et al 1981), or toluene, the solvent in commonly used mounting media such as Fisher Permount (personal observation). The space remaining after dissolution of a bead is not maintained with paraffin embedding as it is with resin embedding. Even after plastic embedding, the resolving power of light microscopes may be inadequate to distinguish such spaces from other spaces found in and between cells. Latex beads are stable in methanol (Van Furth and Diesselhoff-Den Dulk 1980), ethanol. and n-butyl alcohol (Callebaut and Meeussen 1989).     

Freeze-drying of unfixed monolayer cultures for electron microscopic autoradiography

A method has been developed for freezing, drying and embedding of unfixed monolayer cultures for electron microscopic autoradiography (EM ARG). Experimental results showed: a) Aclar 33 C was a more suitable substrate than the plastic of petri dishes, b) cultures pressed rapidly against the polished face of a large copper cylinder chilled in liquid nitrogen had better cellular morphology than did cultures dipped in Freon 12 chilled in liquid nitrogen, and c) cultures embedded in Epon alone had finer extracellular ice spaces and lower background grain densities than did cultures embedded in Epon with 1% silicone. This method has been used to evaluate the effect of fixation on the localization of the neurotransmitter, 3H-gamma-aminobutyric acid (3H-GABA), in neurons of dispersed cell cultures. EM ARG results showed that the neuronal cell bodies and vesicle elements were present in similar numbers in both glutaraldehyde fixed and freeze-dried cultures.     

Freeze-drying of unfixed monolayer cultures for electron microscopic autoradiography

Summary A method has been developed for freezing, drying and embedding of unfixed monolayer cultures for electron microscopic autoradiography (EM ARG). Experimental results showed: a) Aclar 33 C was a more suitable substrate than the plastic of petri dishes, b) cultures pressed rapidly against the polished face of a large copper cylinder chilled in liquid nitrogen had better cellular morphology than did cultures dipped in Freon 12 chilled in liquid nitrogen, and c) cultures embedded in Epon alone had finer extracellular ice spaces and lower background grain densities than did cultures embedded in Epon with 1% silicone.This method has been used to evaluate the effect of fixation on the localization of the neurotransmitter, ³H-gamma-aminobutyric acid (³H-GABA), in neurons of dispersed cell cultures. EM ARG results showed that the neuronal cell bodies and vesicle elements were present in similar numbers in both glutaraldehyde fixed and freeze-dried cultures.     

PLANT MICROTECHNIQUE: SOME PRINCIPLES AND NEW METHODS

Some easily seen structural features of living plant cells are destroyed or badly distorted by most of the common fixatives and embedding media used in plant histology. In stained sections of plant tissues fixed in FAA (formalin-acetic acid-alcohol mixtures) and embedded in paraffin wax, for example, mitochondria and fine transvacuolar strands of cytoplasm are usually not visible. Many structural features such as these can be preserved, however, with suitable fixatives and embedding media. Specifically we recommend fixation in non-coagulant fixatives (e.g., osmium tetroxide, acrolein, glutaraldehyde, formaldehyde) and the use of plastics as embedding media, and we describe in detail a method of fixation in acrolein and embedding in glycol methacrylate polymer. In a wide range of plant specimens prepared in this way, stained sections 1–3 microns thick showed excellent preservation of tissue and cell structures.     

Oriented Embedding of Biological Materials and Accurate Localization for Ultrathin Sectioning

Gelatin capsules with rounded ends clipped off and open ends moistened, affixed to a glass slide and sealed with a 15% gelatin solution are used to embed blocks of tissue in plastic. The surface of the slide serves as an orientation plane for structures of the tissue. The plane end of capsules of polymerized plastic containing no tissue is used in embedding frozen tissue sections. The plastic-infiltrated section is flattened against the capsule end under the weight of a 3/4 inch square of plate glass so that larger sections may be cut and surveyed. Embedding cultured cell monolayers grown on coverslips is accomplished in a comparable manner, but the square of plate glass is not needed as a weight. Block-face localization methods depend on the type of material embedded. With blocks of tissue it is achieved by moistening the face with xylene to develop relief. Thin tissue sections are examined by transmitted light, while cell monolayers are stained on the capsule end with methylene blue.     

Immunogold labeling of luciferase in the luminous bacterium Vibrio harveyi after fast-freeze fixation and different freeze-substitution and embedding procedures

We studied the ultrastructural localization of luciferase on sections of the bioluminescent bacterium Vibrio harveyi by indirect immunogold staining, using a polyclonal antiluciferase antibody and the usual control tests, after chemical fixation or fast-freeze fixation (FFF) followed by different freeze-substitution (FS) procedures and embedding in either Epon or LR White. After liquid fixation with glutaraldehyde and paraformaldehyde and LR White embedding, labeling occurred over the cytoplasm but not over the condensed nucleoid. Epon embedding almost abolished it. FFF-FS considerably improved the morphological preservation and revealed cytoplasmic "patches" with a complex ultrastructure in Epon sections. The preservation was always less good in LR White. The patches were densely labeled, even in Epon sections, after FS in acetone. However, labeling intensity was 3.7 times greater in LR White than in Epon. With both resins, labeling diminished similarly when fixative agents were present in the FS medium. The localization of luciferase in the cytoplasm and particularly in the patches is discussed.     

Histology of plastic embedded amphibian embryos and larvae

Amphibians including the South African clawed frog Xenopus laevis, its close relative Xenopus tropicalis, and the Mexican axolotl (Ambystoma mexicanum) are important vertebrate models for cell biology, development, and regeneration. For the analysis of embryos and larva with altered gene expression in gain-of-function or loss-of-function studies histology is increasingly important. Here, we discuss plastic or resin embedding of embryos as valuable alternatives to conventional paraffin embedding. For example, microwave-assisted tissue processing, combined with embedding in the glycol methacrylate Technovit 7100, is a fast, simple, and reliable method to obtain state-of-the-art histology with high resolution of cellular details in less than a day. Microwave-processed samples embedded in Epon 812 are also useful for transmission electron microscopy. Finally, Technovit-embedded samples are well suited for serial section analysis of embryos labeled either by whole-mount immunofluorescence, or with tracers such as GFP or fluorescent dextrans. Therefore, plastic embedding offers a versatile alternative to paraffin embedding for routine histology and immunocytochemistry of amphibian embryos.     

Value of the K+ salt of carageenan as an agar substitute in routine bacteriological media

The K+ salt of carageenan has no distinct advantages as a gelling agent, but it compared favorably with agar in most of the media tested. The difficulty involved in the preparation of blood plates and the results obtained with this medium prohibit its complete acceptance as a substitute for agar in routine solid media. However, it could be a suitable substitute for agar in all other routine bacteriological media.     

Value of the K+ salt of carageenan as an agar substitute in routine bacteriological media.

The K+ salt of carageenan has no distinct advantages as a gelling agent, but it compared favorably with agar in most of the media tested. The difficulty involved in the preparation of blood plates and the results obtained with this medium prohibit its complete acceptance as a substitute for agar in routine solid media. However, it could be a suitable substitute for agar in all other routine bacteriological media.     

An Embedding Method for Monolayer Cell Cultures for Light and Electron Microscopy

Plastic containers are widely used for monolayer cell culture. In situ embedding, the obvious method of choice for subsequent ultrastructural study, has been achieved by Brinkley et al. (1967) using Epon as a final embedding medium and water soluble acrylates as intermediates. Although the results are satisfactory, the method has two drawbacks: firstly, the water soluble acrylates are difficult to get, and secondly, removal of the plastic container is possible only with blocks already cut off for electron microscopy.     

Ultra-Fast and Optimized Method for the Preparation of Rodent Testicular Cells for Flow Cytometric Analysis

Homogeneity of cell populations is a prerequisite for the analysis of biochemical and molecular events during male gamete differentiation. Given the complex organization of the mammalian testicular tissue, various methods have been used to obtain enriched or purified cell populations, including flow cell sorting. Current protocols are usually time-consuming and may imply loss of short-lived RNAs, which is undesirable for expression profiling. We describe an optimized method to speed up the preparation of suitable testicular cell suspensions for cytometric analysis of different spermatogenic stages from rodents. The procedure takes only 15 min including testis dissection, tissue cutting, and processing through the Medimachine System (Becton Dickinson). This method could be a substitute for the more tedious and time-consuming cell preparation techniques currently in use.     

Fluoroplastic Coverslips for Long-Term Nerve Tissue Culture

Fluoroplastics (Aclar and Teflon FEP) were tested for their suitability as coverslip material for nerve tissue cultures. Coverslips prepared from these plastics have a higher transparency over the near-ultraviolet, visible, and infrared regions of the spectrum than glass coverslips, possess exceptional physical and chemical stability, and provide service-ability over a wide temperature range ≥ -320 to 390 F. For nerve tissue culture, circular 22 mm coverslips were cut from 5 mil sheets of Aclar 22A and 33C, and Teflon FEP 100A, cleaned in concentrated HNO₃, rinsed, sterilized in 85% ethyl alcohol, dried, and coated with collagen. Peripheral and central nervous system cultures from fetal rats and newborn mice were set up on these as well as on glass coverslips, and carried in Maximow double coverslip assemblies. Over a 2 mo period of cultivation, neuronal maturation, tissue organization and fiber myelination occurred on plastic coverslips in essentially the same manner as in sister cultures maintained on glass coverslips. Cultures fixed and embedded in Epon for electron microscopy directly on Aclar or Teflon 100A coverslips were easily separated from the coverslip. Some difficulty was encountered with retraction of the collagen coat from Teflon 100A coverslips, however, and Aclar 22A coverslips were warped by some of the reagents used in embedding for electron microscopy. Aclar 33C was not adversely affected by these reagents, and provided the most suitable base for collagenization and culture maintenance. A variety of cells grew satisfactorily on either the bare or the collagenized surface of this plastic, and separated easily from it following embedment in Epon.     

In situ hybridization of semithin Epon sections with BrdU labelled oligonucleotide probes

Summary We recently described a nonradioactive method for in situ hybridization with 5-bromo-2-deoxyuridine (BrdU) labelled oligonucleotide probes. An antibody to BrdU and immunocytochemistry were used in order to detect the hybridization signal. We have now applied this method to semithin Epon sections, in order to hybridize consecutive sections through single cells with different probes and to stain them with antibodies to neuropeptides. It could be shown that Epon embedding preserves mRNA well. In the present study we used a BrdU labelled synthetic oligonucleotide probe complementary to a fragment of the vasopressin precursor and an antibody to Arg-vasopressin. Vasopressin mRNA was demonstrable in a fraction of the vasopressin immunoreactive neurons in the magnocellular nuclei. In addition some of the magnocellular neurons showed either hybridization or vasopressin immunostaining only, perhaps indicating different stages of synthetic and secretory activity. The method described seems to be a valuable tool for studying synthetic activity in peptidergic neurons on a single cell level. The method might also have potential for in situ hybridization on the electronmicroscopical level.     

In situ hybridization of semithin Epon sections with BrdU labelled oligonucleotide probes

We recently described a nonradioactive method for in situ hybridization with 5-bromo-2-deoxyuridine (BrdU) labelled oligonucleotide probes. An antibody to BrdU and immunocytochemistry were used in order to detect the hybridization signal. We have now applied this method to semithin Epon sections, in order to hybridize consecutive sections through single cells with different probes and to stain them with antibodies to neuropeptides. It could be shown that Epon embedding reserves mRNA well. In the present study we used a BrdU labelled synthetic oligonucleotide probe complementary to a fragment of the vasopressin precursor and an antibody to Arg-vasopressin. Vasopressin mRNA was demonstrable in a fraction of the vasopressin immunoreactive neurons in the magnocellular nuclei. In addition some of the magnocellular neurons showed either hybridization or vasopressin immunostaining only, perhaps indicating different stages of synthetic and secretory activity. The method described seems to be a valuable tool for studying synthetic activity in peptidergic neurons on a single cell level. The method might also have potential for in situ hybridization on the electron-microscopical level.     

Immunoelectron microscopic demonstration of thyroglobulin and thyroid hormones in rat thyroid gland

We have developed a post-embedding immunogold technique for electron microscopic localization and quantitation of thyroglobulin (TG), thyroxine (T4), and triiodothyronine (T3) in rat thyroid. Labeling for TG was located on rough endoplasmic reticulum, Golgi apparatus, exocytotic vesicles, luminal colloid, colloid droplets, and lysosomes, whereas labeling for thyroid hormones was located on luminal colloid, colloid droplets, and lysosomes. We tested different procedures of fixation, dehydration, embedding, polymerization, and immunoincubation to optimize ultrastructural preservation and immunolabeling. Fixation with glutaraldehyde and osmium was possible with retained antigenicity. Dehydration temperature and the choice of embedding resin were the two crucial factors for good immunolabeling. Low-temperature dehydration greatly improved immunolabeling and could be combined with embedding in the methacrylate LR White or the epoxide Agar 100 (equivalent of Epon 812) polymerized at 40-60 degrees C, as the temperature during subsequent embedding and polymerization was of little importance for the immunoreactivity. Labeling on LR White sections was always higher than on Agar 100 sections. Various etching procedures were tested without improved specific labeling. Etching with hydrochloric acid gave nonspecific labeling of certain cell compartments.     

A Silver Impregnation Technique for Normal Axons in the Human Central Nervous System for Celloidin and Epon Sections, with Substitutes for Soft Tap Water

An improved silver technique has been developed for human CNS axons in sections from celloidin blocks that resist impregnation because of prolonged storage in alcohol. This method also gives consistently good impregnation of recently fixed material, and thus is suitable for routine use. Slightly modified, the method is also successful with osmicated Epon embedded sections. The quality of silver impregnation in methods using tap water in the reducing solutions varies in different laboratories. Having established that hard water is essential, substitutes for soft water were sought and found.     

A silver impregnation technique for normal axons in the human central nervous system for celloidin and epon sections, with substitutes for soft tap water

An improved silver technique has been developed for human CNS axons in sections from celloidin blocks that resist impregnation because of prolonged storage in alcohol. This method also gives consistently good impregnation of recently fixed material, and thus is suitable for routine use. Slightly modified, the method is also successful with osmicated Epon embedded sections. The quality of silver impregnation in methods using tap water in the reducing solutions varies in different laboratories. Having established that hard water is essential, substitutes for soft water were sought and found.     

A practical technique to postfix nanogold-immunolabeled specimens with osmium and to embed them in Epon for electron microscopy.

Nanogold is a tiny gold probe, freely diffusible in cells and tissues, and is suitable for pre-embedding immunohistochemistry. However, it is necessary to develop Nanogold to a larger size so that it can be observed by conventional transmission electron microscopy. Silver enhancement is usually used for visualizing Nanogold, but the silver shell produced is unstable in OsO(4) and often becomes invisible after OsO(4) postfixation, which is necessary for good visualization of ultrastructure. We used silver enhancement with silver acetate, followed by gold toning with chloroauric acid, to replace the silver shell with a more stable gold in order to observe Nanogold after osmium fixation and Epon embedding. This technique is applicable to various intra- and extracellular antigens. For correlative observation of immunolabled specimens by light and electron microscopy, specimens adhered to slideglasses were embedded in Epon under non-adhesive plastic film. By heating the Epon sheets after polymerization, these supports were removed without difficulty and provided easy correlative observation.     

Epon-Maraglas Embedment for Electron Microscopy

There can be little doubt that Epon (Luft 1961) is currently the most widely used embedding medium for electron microscopy. While for the most part this embedding material is reliable, it has the disquieting tendency to fail occasionally to polymerize properly for no apparent reason. to counter this problem, several investigators have proposed that epoxide-anhydride ratios be taken into account to arrive at the best balance of ingredients for the final embedding medium (Coulter 1967, Burke and Geiselman 1971, Chang 1973). in an alternative solution, Mollenhauer (1964) suggested that the Epon be mixed with Araldite, and indeed, this combination has achieved some popularity. Epon-Araldite mixtures, however, have a relatively high viscosity in the unpolymerized state; this may slow permeation of tissue specimens.     

The effect of primary fixation with standard postfixation and the duration of hydrolysis on nuclear features in image cytometry.

The effect of three primary fixation procedures, used in the preparation of routine cytological samples: air-drying, Delaunay, and Saccomanno fixation, with postfixation in modified B?hm-Sprenger fixative, on nuclear features as a function of hydrolysis time is reported. Three different cell types: lymphatic cells (tonsil), epithelial cells (buccal mucosa) and mesenchymal cells (uterine myometrium) were used for the study. Our findings show, that generally not all features have the same plateau times as the IOD (integrated optical density), and that many features show different values depending on cell type and fixation method. It is therefore recommended that for any primary fixative used in routine clinical work and for each cell type, the hydrolysis curve for all nuclear features to be used in sample analysis should be established.