Plastic Embedding of thick Celloidin Sections of nerve Tissue

A method is described for mounting Golgi-impregnated and Weigert-stained thick celloidin sections of brain and spinal cord in transparent plastic. Finished mounts have good optical properties and are suitable for macroscopic and microscopic observation. The durability of such preparations makes them superior to similar material prepared by the more conventional methods. Holes of suitable size were cut in matrices of 2.5 × 5 × 3/16 inches Plexiglas. Ward's Bio-plastic was used to form a base for the holes and also as the embedding medium for the sections. Plate glass formed a working substrate and gave a polished surface to the plastic base and later to the top of the preparation. For Golgi material (200μ) the celloidin was removed by dioxane. A dioxane-plastic bath preceded plastic embedding. For Weigert material (30-40μ) celloidin was not removed due to fragility of sections. Prior to plastic embedding, they were subjected first to benzol and then to a benzol-plastic bath.     

The Rapid Preparation of Frozen Tissue Sections

The essential feature of this procedure involves the rapid freezing of the tissue following excision and keeping it frozen until the desired chemical or fixative has been applied. For freezing, either carbon dioxide or liquid air is used, as desired. The microtome knife is thoroughly cooled by taping blocks of dry ice to its surface. The cut sections, still frozen, are manipulated by a camel's hair brush so that they lie flat upon the knife. They are then transferred to a slide by a special section lifter. This has the form of a double-bottomed scoop packed with dry ice. Thus the section remains frozen while it is transferred to a clean microscope slide held at an angle above a Coplin jar of the desired reagent. The sections must be immersed just prior to melting. They curl and do not adhere to the slide if still rigidly frozen, and are distorted if immersed after melting.

With this technic sections showing a minimum of cellular distortion may be obtained. Consequently, it facilitates the use of many cytological technics, chemical tests, and enzymatic studies, such as the Gomori technics, on a variety of tissues.     

Rapid Preparation of Thick Sections of Fleshy Plant Tissues

Methods are described for permanent micro-slide preparations of soft, large-celled plant tissues such as ripe fruit. Thick sections (200-800 [t) cut on a sliding microtome are aspirated in an aqueous killing agent; after fixing and washing, the sections are dehydrated and cleared in an alcohol-xylene series. Infiltration with 20, 30, and 40% solutions of mountant prior to mounting the sections is necessary to avoid too abrupt changes in the cleared tissues. Several staining methods have been successfully used for different purposes. The final preparations showed nearly perfect preservation of intact cells and intercellular spaces in their 3-dimension-al structure.     

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.     

A Simple Device to Help Re-Embed Thick Plastic Sections

The removal of epoxy capsules cast on glass slides is facilitated by 1) partial polymerization and brief exposure to elevated temperature, and 2) use of a slide holder to support the hot slides and reduce the chance of breakage. With this procedure, plastic sections routinely dried onto glass slides are available for re-embedding and subsequent thin sectioning.     

A Plastic Embedding Technique for Analyzing Fluorescent Dextran-Amine Labelled Neuronal Profiles

A plastic embedding technique employing fluorescently labelled dextran-amines is described. After application of tracer to cut nerves and appropriate transport time, animals were fixed in paraformaldehyde. Subsequently their brains were dissected, heads and brains were dehydrated, embedded in methacrylate and sectioned serially on a rotary microtome. Plastic sections allow high resolution of single neuron profiles and complete serial reconstruction of un-distorted sections, including embryos with large amounts of yolk. In conjunction with whole mount analysis and double labelling, this technique can accurately reveal the spatial relationships of nerve components throughout development.     

An Improved Method for Embedding Hard Tissue in Poeymethyl Methacrylate

An improved routine method for embedding tissue, especially hard tissue, in poly-methyl methacrylate (pMMA) is described. The improvements were: the final dehydration step before MMA infiltration was performed with methanol in a Soxhlet apparatus; the stabilizer hydroquinone was not extracted from the monomer (MMA), and more important, the commonly used polymerization initiator, benzoyl peroxide (bpo), was replaced by the initiator, bis (4-tert-butylcyclohexyl)peroxydicarbonate (bbpd). Bbpd is preferred to bpo because it is not explosive, far less is needed and it has a suitable half life. Moreover, bbpd, as obtained from the manufacturer, needs no further purification, in contrast to bpo. Temperatures during bbpd initiated polymerization did not exceed 48 C. In bbpd initiated pMMA, bubbles were almost never generated.     

Carbowax Embedding for Obtaining Thin Tissue Sections and Study of Intracellular Lipids

Carbowax, a water soluble wax, as an embedding agent is a valuable adjunct to the armamentarium of the tissue technologist. This report is intended to supplement previous publications on the use of Carbowax and to indicate die necessity for preheating and variation of Carbowax mixtures according to the climate.

Carbowax embedding provides an easy means for obtaining tissue sections 1 to 3 μ in thickness either with or without previous exposure to fat solvents. These sections are admirably suited for cytological study, particularly of intracellular lipoids.     

A Method for the Preparation of Serial Thick Sections of Plastic-Embedded Plant Tissues

A procedure is described in which thick sections (2-10μ or more) of plastic-embedded plant tissues are mounted in serial order on slides for use in routine light microscopy. Sections are cut with a steel knife on a rotary microtome while the block and blade are bathed with 40% alcohol. The cut sections are placed, in order, in 50% alcohol in the small wells of modified plastic trays where they become flat, pliable and suitable for subsequent handling. Sections remain separate and in correct order in the trays while they are stained, washed, and prepared for final mounting on slides. Mounting involves a simple and rapid procedure of transferring the sections to a slide and heating first on a 70-75 C hot plate (to slowly evaporate the water around the section and to partially affix the section) and then on a 100 C hot plate. This second heating ensures adhesion when xylene-base mounting media, which tend to loosen weakly adhered plastic from the slides, are used. The technique of staining the sections loose provides the following advantages: (1) the problems of section loss and entrapment of stain between section and slide during staining are eliminated, (2) relatively high staining temperature, akalinity, and alcohol concentration of the stain solvent (all of which promote loosening of pm-affixed sections from slides during staining) is allowed, and (3) staining is more even and selective. The procedure has been found to be reliable and fast enough to be of value in a significant variety of routine light microscope studies.     

Thin Histological Sections Prepared from Large Thick Sections: a New Technique

A method is described for obtaining thin (1 μm) sections for light microscopy from large area thick (100 μm) sections of low viscosity nitrocellulose embedded specimens of human spinal osteoligamentous material.     

Thick Sections for Embryology

Night blue will stain the mast cells of rat, mouse and hamster selectively if alcohol differentiation is controlled. The technical steps are: Dewax paraffin sections with xylene, 2 changes; air dry; 2% Na₂SO₄, 3-5 sec; 0.5% night blue in 10% ethanol, 1 hr at 60°C; rinse in water; 9% HNO₃, 15 sec; water 1-5 min; 70% ethanol, 2 changes, 30 sec each; wash; 0.01% safranin, 3-5 sec; rinse, blot, air dry, mount in synthetic resin. A clear orthochromatic stain of the mast-cell granules occurs. Acid fixation prevents the staining reaction.     

Fluorescence of Plastic Embedded Tissue Sections After Curcumin Staining

The natural dye, curcumin (C.I. 75300) from turmeric, is obtained from the roots of Curcuma longa (Lillie 1977). Curcumin has scarcely been applied for histological work, and its fluorescence seems to have been overlooked. During the course of studies on fluorescent aluminum complexes (Del Castillo et al. 1987) we realized that this dye induces a green fluorescence of chromatin (Stockert et al. 1989). In this note we describe the fluorescence reaction of curcumin on semithin sections of olastic embedded tissues.     

A Disposable Plastic Box for Paraffin Embedding

A sheet of cellulose acetate about 0.01 inch thick is clamped over a mold, heated to softness by an electric heater and drawn down over the mold by means of a vacuum. When cooled, the sheet, now formed into embedding boxes, is removed from the clamp. Boxes so made are inexpensive enough to be disposable but can be reused, since the sides of the boxes are sloped to allow easy removal of the paraffin block.     

A Cytochemical Technique for Demonstration of Lipids,Polysaccharides and Protein Bodies in Thick Resin Sections

An effective cytochemical technique for the simultaneous demonstration of lipids, polysaccharides and protein bodies in the same section from the tissue embedded in Epon 812 is described. Thick sections of peanut cotyledon are used for a typical sample according to the following procelures. Firstly, PAS reaction: (1) Oxidize sections in 0.5% periodic acid in 0.3% nitric acid for 10 min, (2) Wash in running water for 1–2 min and then pass through distilled water, (3) Stain in Schiff's reagent for 30 min, (4) Wash in sodium metabisulfite 3 times, 2 min for each time, (5) Wash in running water for 5 min and then pass through distilled water. Secondly, Sudan black B staining: (1) Rinse section in 70% ethanol for 1-2 min, (2) Stain in fresh 1% Sudan black B in 70% ethanol for 30–60 min at 40–60℃, (3)Rinse in 70% ethanol for 1 min and then in distilled water. Thirdly, Coomassie brilliant blue R staining: (1) Rinse sections in 7% acetic acid for 1–2 min, (2) Stain in I% Coomassie brilliant blue R in 7% acetic acid for 20 min at 60℃, (3) Differentiate in 0.1% acetic acid for I min, (4) Rinse in lunning water for 5 min and then pass through distilled water, (5) Dry at room temperature or in oven, 40℃. The dry sections mount in glycerin-gelatin. After the above three step staining, the three main compounds of the cell can be stained simultaneously. Starch grains and cellulose cell wall take cherry red colour, lipids appear in black, protein bodies are blue. The sealed slides can be kept permanently.     

Rapid Otan Method for Localizing Unsaturated Lipids in Lung Tissue Sections

The OTAN treatment, which is the only histochemical method available at present for the simultaneous localization of hydrophobic and hydrophilic unsaturated lipids in tissue sections, requires unduly long exposure to O₃O₄ and use of free-floating sections, which makes handling the sections difficult and often results in their loss or damage. Simple modifications using O₃O₄ treatment at 37 C and slide-mounted sections eliminate the practical drawbacks of the existing method and provide as good or better localization in less than one-eighth of the time. The modified method is applicable to fixed as well as fresh frozen tissues.     

A Simple Two-Dye Basic Stain Facilitating Recognition of Mitosis in Plastic Embedded Tissue Sections

Semithin sections of buccal and palatal mucosa fixed in 2.5% glutaraldehvde followed by 1% osmium and embedded in Durcupan (an araldite-baaed resin) were stained with 2% malachite green in 50% ethanol at 80 C and poststained in 0.05% crystal violet in Sorensen's phosphate buffer (pH 6.4) at 45 C Nuclear envelopes and chromatin stain vivid purple in contrast to the surrounding green cytoplasm and cell borders. Chromosomes of dividing cells stain bluish violet. Nucleoli, depending on their level in the epithelium, stain differing shades of greenish blue. The distinct and differential staining of each of these components facilitates recognition of mitoses in oral epithelium, where the small sice and crowding of cells in the proliferative compartment renders more conventional stains for plastic sections inadequate.     

Gelatin Embedding of Central Nervous System Tissue Improves the Quality of Vibratome Sections

We have developed a procedure for Vibratome (Oxford Laboratories) sections that is particularly valuable for providing uniformly thick, well preserved CNS tissue sections for morphometric applications.     

Gelatin Embedding of Central Nervous System Tissue Improves the Quality of Vibratome Sections

We have developed a procedure for Vibratome (Oxford Laboratories) sections that is particularly valuable for providing uniformly thick, well preserved CNS tissue sections for morphometric applications.     

Nongraded Dehydration and Low-Pressure Infiltration for Rapid Celloidin Embedding of Brain Tissue

Various ways of shortening single steps in the celloidin process have been combined to form a routine method which may be completed, for tissues of average size, within a week following fixation. Fixed, washed tissue slices 5 mm thick are dehydrated in 1 or 2 changes of absolute ethanol and acetone, 1:1. This requires 24 hr in an incubator at 37 C, or 12-16 hr if a magnetic stirrer is used. After ether-alcohol for 4 hr. the tissues are transferred to 5% celloidin and infiltrated in a vacuum desiccator attached to a filter pump. When the volume of celloidin is reduced to half the original amount (about 2 hr), the tissues are removed from the infiltrating fluid and embedded in 10% celloidin. The blocks are hardened in chloroform and cleared by suspending them in 2 or 3 changes of terpineol agitated by a magnetic stirrer. Sections are cut in terpineol, using any type of microtome. After washing in 95% alcohol, they are mounted on albumenized slides for staining.