An improved Timm sulphide silver method for light and electron microscopic localization of heavy metals in biological tissues.

Modifications of the Timm sulphide silver method for the demonstration of heavy metals are described. To improve the structural preservation of the tissues perfusion with a glutaraldehyde fixature is employed before perfusion with the sodium sulphide solution. For the subsequent staining for light and electron microscopy, procedures for plastic embedding, paraffin embedding and cryostat sectioning are presented. Examples from several tissues are shown, including the pituitary, pancreas, intestine, tongue, kidney, testis and brain. The staining of autolytic, postmortal human brain tissue is demonstrated.     

An improved timm sulphide silver method for light and electron microscopic localization of heavy metals in biological tissues

Summary Modifications of the Timm sulphide silver method for the demonstration of heavy metals are described.To improve the structural preservation of the tissues perfusion with a glutaraldehyde fixative is employed before perfusion with the sodium sulphide solution. For the subsequent staining for light and electron microscopy, procedures for plastic embedding, paraffin embedding and cryostat sectioning are presented. Examples from several tissues are shown, including the pituitary, pancreas, intestine, tongue, kidney, testis and brain. The staining of autolytic, postmortal human brain tissue is demonstrated.     

Histochemical and immunohistochemical protocols for routine biopsies embedded in Lowicryl resin

We describe a novel method that allows reliable detection of in situ hybridization signals in thin sections of plastic embedded embryos. Sections from plastic embedded embryos are thinner and have superior histological quality compared to paraffin, gelatin, agarose embedded sections or cryosections; however, plastic resin traditionally has not been used as an embedding medium following in situ hybridization because of loss of signal. When signal is detected with alkaline phosphatase and NBT/BCIP, the resulting colored precipitate is subject to fading when samples are exposed to organic compounds. The colored precipitate can be redeposited by repeating the NBT/BCIP reaction following plastic sectioning. This recolorization shows no loss of specificity, because signal is detected only where the anti-digoxigenin/alkaline phosphatase conjugated antibody is bound to the riboprobe. Strong signals can be detected without recolorization; however, weaker signals require the recolorization step. This novel method of re-depositing colored precipitate after processing and sectioning allows accurate determination of the location of gene expression and study of this expression in high quality histological sections of early chick embryos.     

Improvements in histological quality and signal retention following in situ hybridization in early chick embryos using plastic resin and recolorization.

We describe a novel method that allows reliable detection of in situ hybridization signals in thin sections of plastic embedded embryos. Sections from plastic embedded embryos are thinner and have superior histological quality compared to paraffin, gelatin, agarose embedded sections or cryosections; however, plastic resin traditionally has not been used as an embedding medium following in situ hybridization because of loss of signal. When signal is detected with alkaline phosphatase and NBT/BCIP, the resulting colored precipitate is subject to fading when samples are exposed to organic compounds. The colored precipitate can be redeposited by repeating the NBT/BCIP reaction following plastic sectioning. This recolorization shows no loss of specificity, because signal is detected only where the anti-digoxigenin/alkaline phosphatase conjugated antibody is bound to the riboprobe. Strong signals can be detected without recolorization; however, weaker signals require the recolorization step. This novel method of re-depositing colored precipitate after processing and sectioning allows accurate determination of the location of gene expression and study of this expression in high quality histological sections of early chick embryos.     

Embedding, serial sectioning and staining of zebrafish embryos using JB-4 resin

Histological techniques are critical for observing tissue and cellular morphology. In this paper, we outline our protocol for embedding, serial sectioning, staining and visualizing zebrafish embryos embedded in JB-4 plastic resin-a glycol methacrylate-based medium that results in excellent preservation of tissue morphology. In addition, we describe our procedures for staining plastic sections with toluidine blue or hematoxylin and eosin, and show how to couple these stains with whole-mount RNA in situ hybridization. We also describe how to maintain and visualize immunofluorescence and EGFP signals in JB-4 resin. The protocol we outline-from embryo preparation, embedding, sectioning and staining to visualization-can be accomplished in 3 d. Overall, we reinforce that plastic embedding can provide higher resolution of cellular details and is a valuable tool for cellular and morphological studies in zebrafish.     

Glycol Methacrylate in Light Microscopy a Routine Method for Embedding and Sectioning Animal Tissues

Glycol methacrylate (GMA), a water and ethanol miscible plastic, was introduced to histology as an embedding medium for electron microscopy. This medium may be made soft enough for cutting thick sections for routine light microscopy by altering its composition. A procedure for the infiltration, polymerization, and sectioning of animal tissues in GMA for light microscopy is presented which is no more complex than paraffin techniques and which has a number of advantages: (I) The GMA medium is compatible with both aqueous fixatives (formaldehyde, glutaraldehyde, Bouin's, and Zenker's) and non-aqueous fixatixes (Carnoy's, Newcomer's, ethanol, and acetone). (2) Undue solvent extraction of the tissue is avoided because adequate dehydration occurs during infiltration of the embedding medium. Separate dehydration and clearing of the tissue prior to embedding is eliminated. (3) When polymerized, the supporting matrix is firm enough that hard and soft tissues adjacent to one another may be sectioned without distortion. (4) Thermal artifact is reduced to a minimum during polymerization because the temperature of the tissue may be maintained at 0-4 C from fixation through ultraviolet light polymerization of the embedding medium. (5) Shrinkage during polymerization of the embedding medium is minimized by prepolymerization of the medium before use. (6) Sections may be easily cut using conventional steel knives and rotary microtomes at a thickness of 0.5 to 3.0 microns, thus improving resolution compared with routinely thicker paraffin sections. (7) The polymerized GMA medium is porous enough so that staining, auto radiography, and other histological procedure are done without removal of the embedding medium from the sections. A list of these stains and related procedures are included. (8) Enzyme digestion of ultra thin sections of tissue embedded in GMA is common in electron microscopic cyto chemistry. me same digestion techniques appear compatible with the thicker seaions used in light microscopy.     

Double Embedding Broad Thin Leaves in Paraffin

Paraffin pellets were melted in 24 × 24 × 5 mm stainless steel base molds. Specimens of leaves, 18 × 18 mm, were fixed, dehydrated and infiltrated with paraffin. Two specimens were transferred into molten paraffin on their laminar surfaces in a base mold and moved quickly onto a cold surface to cast them in a shallow block of paraffin. Each block was then scored with a razor blade, broken into two primary blocks, and trimmed to 20 × 9 mm with 5 mm flat edges. Each primary block was immersed upright on its long edge in a 22 × 22 × 20 mm Peel-A-Way® embedding mold containing molten paraffin. The leaf edge was held centrally in the mold while moving the double embedment onto a cold surface. In this secondary block, the leaf specimen stood perpendicular to the sectioning surface in perfect orientation for transverse ribbon sectioning. The two phases of paraffin bonded well.     

N-butyl Methacrylate and Paraffin as an Embedding Medium for Light Microscopy

A method of tissue embedding using n-butyl methacrylate and paraffin is described. Following alcohol dehydration and infiltration with the methacrylate monomer, tissues are embedded in gelatin capsules in a mixture consisting of 3.5 g of paraffin for each 10 ml of methacrylate. Benzoyl peroxide (0.2 g for each 10 ml of monomer) is added as the catalyst and the methacrylate polymerized in a 50 C oven for 18-24 h. Following polymerization the block is trimmed and embedded in paraffin to provide a firm support during sectioning. A water trough attached to the microtome knife is essential to facilitate the handling of sections and ribbons. For serial sections a mixture of equal weights of beeswax and paraffin is used to make the sections adhere to each other. Usual staining procedures can be used since the embedding medium is readily soluble in xylene.     

N-Butyl methacrylate and paraffin as an embedding medium for light microscopy.

A method of tissue embedding using n-butyl methacrylate and paraffin is described. Following alcohol dehydration and infiltration with the methacrylate monomer, tissues are embedded in gelatin capsules in a mixture consisting of 3.5 g of paraffin for each 10 ml of methacrylate. Benzoyl peroxide (0.2 g for each 10 ml of monomer) is added as the catalyst and the methacrylate polymerized in a 50 C oven for 18--24 h. Following polymerization the block is trimmed and embedded in paraffin to provide a firm support during sectioning. A water trough attached to the microtome knife is essential to facilitate the handling of sections and ribbons. For serial sections a mixture of equal weights of beeswax and paraffin is used to make the sections adhere to each other. Usual staining procedures can be used since the embedding medium is readily soluble in xylene.     

Science and art in preparing tissues embedded in plastic for light microscopy, with special reference to glycol methacrylate, glass knives and simple stains.

Plastic embedding preserves tissue structure much more faithfully than does paraffin. Acrylic polymerization is innocuous to dye-binding groups in sections. The water solubility of glycol methacrylate monomer and the hydrophilic properties of the polymer allow for convenience in dehydration and for versatility in staining sections. Five years of experience with glycol methacrylate (GMA) embedding for light microscopy is summarized. Methods for purifying GMA monomer are cited. Procedures for fixing, dehydrating, embedding, polymerizing, sectioning and staining, using GMA, are explained. A method is provided for making glass knives long enough to cut large blocks. Simple, reliable, quick staining methods are outlined. When compared with paraffin, GMA offers opportunities for simpler, quicker procedures and yields sections of superior quality, greater information content, and less distortion.     

A Simplified and Convenient Method for the Double-Embedding of Tissues

A method for embedding tissues with a celloidin-paraffin combination is presented. The essential features of the process depend upon (1) a thorough infiltration of the specimen with celloidin of low concentration, and (2) the subsequent impregnation of both the specimen and the celloidin with paraffin.

The methods for sectioning, and the removal of the embedding agent are given.

The chief advantages of this method are: the preservation of all of the advantages of celloidin embedding but with a great saving of time, and greater convenience of storage; the cutting of thin sections (2μ for many types of tissues); it is useful for embedding specimens for which neither pure paraffin nor pure celloidin are entirely satisfactory, i.e. those containing tissues differing in density.     

Histochemical demonstration of heavy metals

Summary The three steps of the sulphide silver method have been examined: 1) Transformation of metals to metal sulphides; 2) Fixation and embedding or freezing of the tissue for sectioning; and 3) Deposition of metallic silver on the metal sulphides in a physical developer. Based on the results, a revised method is described and discussed. It is particularly important 1) To maintain a sufficient but low concentration of sulphide ions during the perfusion; 2) To avoid using oxidating or acid fixatives; 3) To ensure low temperatures while embedding in paraffin or during polymerization of Epon; and 4) to use a slow-acting physical developer. Examples of the metal sulphide pattern from various tissues are presented.     

Lucifer yellow, neuronal marking and paraffin sectioning

An unusual procedure with the dye lucifer yellow has provided stable neuronal marking that survives paraffin embedding and sectioning. Lucifer yellow CH was dissolved in an electrolyte containing formaldehyde and injected into the large interneurons of a cricket. Intense fluorescence in the axoplasm was retained even after conventional histological procedures.     

Science and Art in Preparing Tissues Embedded in Plastic for Light Microscopy, with Special Reference to Glycol Methacrylate, Glass Knives and Simple Stains

Plastic embedding preserves tissue structure much more faithfully than does paraffin. Acrylic polymerization is innocuous to dye-binding groups in sections. The water solubility of glycol methacrylate monomer and the hydrophilic properties of the polymer allow for convenience in dehydration and for versatility in staining sections. Five years of experience with glycol methacrylate (GMA) embedding for light microscopy is summarized. Methods for purifying GMA monomer are cited. Procedures for fixing, dehydrating, embedding, polymerizing, sectioning and staining, using GMA, are explained. A method is provided for making glass knives long enough to cut large blocks. Simple, reliable, quick staining methods are outlined. When compared with paraffin, GMA offers opportunities for simpler, quicker procedures and yields sections of superior quality, greater information content, and less distortion.     

Lucifer Yellow, Neuronal Marking and Paraffin Sectioning

An unusual procedure with the dye lucifer yellow has provided stable neuronal marking that survives paraffin embedding and sectioning. Lucifer yellow CH was dissolved in an electrolyte containing formaldehyde and injected into the large intemeurons of a cricket. Intense fluorescence in the axoplasm was retained even after conventional histological procedures.     

Zur Verwendung von 2-Hydroxyethyl-Methacrylat (GMA) als Einbettungsmedium bei histologischen Untersuchungen in der Phytopathologie

The use of 2-hydroxyethyl-methacrylate (GMA) as embedding medium for histological investigations in phytopathology A new plastic embedding technique is described for subsequent thin sectioning of plant tissues. In comparison to the paraffinmethod the GMA polymerization system is less time consuming. The excellent preservation of well-fixed tissue is fully asserted, as the embedding medium is not removed from the sections. In lightmicroscopic studies convincing results were obtained with different staining procedures; specific evidence for polysaccharides, pectine and nucleic acids was carried out with thin sections of 2-8 μm thickness, also by fluorescence microscopy. The GMA-embedding technique seems to be of value for various histological investigations in phytopathology.     

Diethylene Glycol Distearate Embedding and Ultramicrotome Sectioning for Light Microscopy

Diethylene glycol distearate can be used as an embedding medium for light microscopy. Two infiltration changes of about 6 hr each in the melted wax (melting point 47-52 C) are required before the final embedding which is done in 00 gelatin capsules for sectioning in the ultramicrotome by the procedure used in electron microscopy. Serial sections 1-2 μ thick can be cut without difficulty. No cooling devices are necessary for trimming and sectioning at laboratory temperature. Sections rarely become detached from the slides. The staining characteristics of the tissues are the same as when embedded in paraffin. For fluorescence microscopy, essentially the same procedure is followed. Tissues are not distorted and the intracellular structures are well preserved.     

Neurites and growth cones in the chick embryo. Enhanced tissue preservation and visualization of HRP-labeled subpopulations in serial 25-microns plastic sections cut on a rotary microtome

Study of axonal guidance in developing vertebrates has been hindered by an inability to readily visualize individual growth cones, determine the neuronal population from which they originate, trace their trajectories, and discern their interactions with their embryonic environment. We report a method that combines plastic embedding and serial sectioning with horseradish peroxidase labeling of subpopulations of neurons in the chick embryo. This method labels individual neurites from the soma to the tip of the growth cones, allowing their trajectory to be inferred and their identity to be determined by the position of the somata. As sections are up to 25 micron thick, entire growth cones can often be visualized without laborious reconstruction. Tissue preservation is much better than with similar material embedded in paraffin. Sections are cut relatively quickly using a steel knife on a standard rotary microtome and are suitable for subsequent electron microscopy.     

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.     

A Squeeze-Out Method,Using Pliers,for Removing Hardened Blocks from Polyethylene Capsules

The original BEEM capsule #1000 (Better Equipment for Electron Microscopy, P.O. Box 132, Jerome Station, Bronx, NY 10469) is now widely used as a casting mold in plastic embedding. This polyethylene capsule has proven popular with many electron microscopists because it provides a preshaped truncated pyramid casting that requires minimal trimming prior to sectioning. A disadvantage of the plastic capsule is that it must be either laboriously slit or partially cut away to allow removal of the hardened block; or, one may use a special, rather costly press designed for such removal.