Epoxy Embedding Research Articles

An epoxy resin embedding technique for large objects.

Undecalcified heads in sizes up to that of a mallard are embedded, sectioned (10μ), and stained according to a modification of Luft's procedure (1961). Polymerization time is prolonged by keeping the resin mixture at 4 C or by omission of the accelerator. Under these conditions embedding takes about a month. The transparency of the blocks facilitates exact orientation of the object with respect to the sectioning plane, and milled reference grooves perpendicular to the sectioning plane permit exact reconstruction. Staining and mounting procedures are described. The technique has proved useful for exact determination of the spiritual disposition of the components of an object at a level between the microscopic and the anatomical.

Ultraviolet microscopy at wavelengths below 240 nm

SummaryModification of the techniques used for ultraviolet microscopy have allowed extension of the usable wavelength range down to about 220 nm. The 150 W xenon lamp in a Leitz ultraviolet microscope was replaced with a 1000 W xenon‐mercury lamp. A special low gelatin content film was employed and a new epoxy embedding medium of low UV absorbance was formulated.

Epoxy embedding, sectioning and staining of plant material for light microscopy.

By using a formula which gives a relatively soft epoxy embedding medium, it is possible to cut sections of plant material with a sliding microtome equipped with a regular steel knife. Blocks having a cutting face of 10 × 10 mm, giving sections of 4–10 μm, can be used. Tissues are fixed in Karnovsky's fluid, postfixed in 1 or 2% OsO4, embedded in Spurr's soft epoxy resin, Araldite, or Epon mixtures. 5% KMnO4, followed by 5% oxalic acid, then neutralized in 1% LiCO3, are used to mordant the sections. Some of the stains used are Mallory's phosphotungstic acid-hemotoxylin, acid fuchsin and toluidine blue, or toluidine blue. Mounting is done with whichever soft epoxy resin was used in casting the blocks.

Quantitative electron probe analysis of soft biologic tissue for electrolytes.

Electron probe analysis of soft biologic tissue requires the use of a tissue preparation that (a) presents a flat surface of the specimen to an electron beam in a vacuum, (b) can withstand the rigors of a vacuum, (c) can withstand damage by an electron beam and (d) causes no redistribution of the elements under study. A tissue preparation which also does not involve the use of aqueous solutions for any step of the preparation has been used for the examples of electron probe analyses presented here. Tissue is prepared by quick freezing at about –196°C, drying in a vacuum at between –60°C and –85°C, fixing with osmium tetroxide vapor and embedding in a low Cl epoxy. Calibration of the electron probe signals is accomplished with prepared solutions of 20% albumin and known quantities of electrolytes. Cell morphology is well preserved to the submicron level, but counting rates and signal to background ratios for the x-ray signals are low. With a 10-kV, 50-na electron beam, the spatial resolution is comparable to that of light microscopy with detection limits for K, Cl and Na of a few milliequivalents per liter. Analysis of frog skeletal muscle fibers yielded concentrations of 122.4 ± 2.8 (s.e.) mEq/liter for K, 10.5 ± 0.7 mEq/liter for Cl and 9.2 ± 1.0 mEq/liter for Na as the averages of nine fibers from one muscle. The Ca content in a discontinuous extracellular layer in frog skin was found to be as high as 4-5 M with a Ca:P ratio of an apatite when referenced to a natural fluoroapatite crystal.

A new technique for separation of coverglass substrate from epoxy-embedded specimens for electron microscopy

A simple and reproducible new technique for growing cells on coverglasses for subsequent epoxy embedding has been developed. Cleaned coverglasses are lightly sprayed with Teflon from a pressurized can and then heated at 250°C for 30 minutes. After sterilization, the treated coverglasses are ready for growing cells. When the growth reaches optimum, the monolayer cells attaching to the coverglass are fixed, dehydrated, and embedded into Epon or other plastics. Clean separation between the coverglass and embedding plastics can be assured by sudden cooling to liquid nitrogen temperature. In addition to monolayer cells, the technique is also applicable to the processing of frozen sections, fresh imprints, ascites cells, and squash preparations as well as blood and bone marrow smears. It allows preselection of cell components or cells for correlated light and electron microscopy and multiple sampling from a single culture flask or dish for time sequence or similar experiments. These and other advantages make the technique useful in modern electron microscopy.

Differentiation of Ribosomes and Beta Glycogen in Canine Myocardium

During the ultrastructural examination of the canine myocardium, it became apparent that the positive identification of specific granules as either ribosomes or β glycogen was very difficult in sections stained with lead citrate and uranyl acetate. In the normal adult canine myocardium ribosomes are not a prominent organelle, but in certain conditions, free ribosomes may become greatly increased in number. Unlike some other species, glycogen and ribosomes are very nearly the same size (200-250 Å) in the canine myocardium. Following glutaraldehyde fixation and epoxy resin embedding there is considerable variation in electron density of glycoen (Fig. 1).In an attempt to differentiate glycogen from free ribosome particles, myocardial samples from four normal dogs and six dogs with experimental right ventricular hypertrophy were processed 1) according to routine procedure, 2) following autolysis, 3) following diastase digestion and 4) by the periodic acid silver proteinate method of Theiry (J. Microscopie 6:986, 1967).

The connective tissue ground substance in the mouse uterine cervix: an electron microscopic histochemical study.

AbstractSections of uterine cervix from ovariectomized mice treated with relaxin (R) or estradiol benzoate (EB) alone or EB and R in combination were exposed to dialyzed iron solutions prior to epoxy embedding. Control mice were untreated or received the hormone vehicles. Prior to iron staining, some sections were exposed to: (1) 0.05% testicular hyaluronidase for four hours at 37°C; or (2) acidified methanol for four hours at 60°C. Ultrastructurally, the uterine cervical walls from control mice and those treated with R contained densely packed bundles of collagenous fibrils and cells that resembled fibrocytes with few organelles. Except for spurious intracellular iron deposits, there was no iron staining of extracellular components.Fibroblasts of cervices from mice treated with EB or EB and R were larger with highly developed endoplasmic reticulum and Golgi components. Small cytoplasmic vesicles were present close to the cell surface and each contained a thin inner rim of material. The stroma was characterized by widely dispersed bundles of collagenous fibrils in a greatly expanded cervical wall where there was extracellular iron staining that was prominent near cell surfaces and between individual fibrils within an amorphous material. Brief digestion with testicular hyaluronidase resulted in a reduction of extracellular iron staining, although deposits remained adjacent to cell surfaces, between fibrils and within cytoplasmic vesicles. Acidic methylation caused severe tissue damage so that an assessment of iron deposition in thin sections was impossible.

Embedding free-floating cells in stained agar for rapid scanning and subsequent ultramicrotomy.

Methods we have previously used for epoxy embedding of suspended tissue culture cells, white blood cells and oral mucosal scrapings did not provide a simple means for selecting the desired specimens and facilitating their subsequent handling through embedding in an epoxy resin. We have therefore modified the agar embedding technique previously recommended for the processing of microorganisms by Kellenberger et al. (1958).

Leaf wax fine structure and ontogeny in Eucalyptus demonstrated by means of a specialized fixation technique

SUMMARYThe epidermal cells of Eucalyptus leaves are not well preserved by the conventional sectioning methods used for electron microscopy. More satisfactory preservation results from the use of an in vivo perfusion technique using glutaraldehyde or glutaraldehyde‐formaldehyde mixtures. This technique preserves wax on the cuticle surface, rendering it insoluble in the organic solvents used for dehydration and embedding in epoxy resins. Using this method, no direct connection is apparent between leaf wax, cuticle, cell wall and cytoplasm. The Golgi bodies appear to produce vesicles which migrate only in the direction of the outer cell wall, and which might liberate wax precursors into the wall, from where they could diffuse through cuticular lamell to polymerize or oxidize at the leaf surface as waxes of characteristic morphology.

Transmission Electron Microscopy of Green Sheet Ceramic Specimens Prepared by Ultramicrotomy

It is often desirable to make high-magnification observations of the internal structure of green sheet ceramic. Scanning electron microscopy and replication microscopy have been unsatisfactory in studying the structure of green sheet ceramic. The feasibility of another technique, that of combining ultramicrotomy with transmission electron microscopy (TEM), seemed, therefore, worth investigating. Mullite and alumina green sheets were studied.Narrow (1 × 10mm) strips of green sheet ceramic were cut and mounted in “Beem” microtome capsules. A mixture of Cargille NYSEM epoxy embedding materials was prepared and poured into the capsules, care being taken to keep the specimen centered, especially at the tapered end of the capsule.

Electron probe analysis of freeze-substituted, epoxy resin embedded tissue for ion transport studies in plants

A new technique is described to prepare plant material for electron probe analysis. Root segments 1 mm in length were frozen at-170°, freeze-substituted with anhydrous ether at-30° and infiltrated with Spurr's low-viscosity epoxy resin embedding medium at low temperatures. Sections 1 and 2μ thick were cut anhydrously using hexylene glycol in the ultramicrotome trough, mounted on the polished surface of a Be disc and vacuum coated with 150-200 Å aluminum.The new technique allows retention of water-soluble ions at the original sites in the tissue and is superior to cryostat sectioning in spatial resolution of electron probe analysis and in the preservation of cellular structures.The lateral transport of K(+) into the xylem of corn roots has been successfully studied by electron probe analysis of freeze-substituted, epoxy resin embedded material.

Use of tissues embedded in epoxy resin for routine histological examination of renal biopsies.

The production of 0.4mu thick, full-length sections from renal tissue taken by needle biopsy is achieved by formalin and osmic acid fixation and embedding in epoxy resin as for ultramicrotomy. The marked improvement in histological clarity by using this technique is illustrated by comparison with material embedded in paraffin.

P-(acetoxymercuric) aniline diazotate, a reagent for visualizing the naphthol AS-BI product of acid hydrolase action at the level of the light and electron microscope.

Diazotized acetoxymercuric aniline in a postcoupling procedure for β-glucuronidase and acid phosphatase using naphthol AS-BI β-d-glucosiduronic acid and naphthol AS-BI-phosphoric acid respectively as substrates has yielded excellent 2-µ sections from Araldite-embedded specimens for light microscopy and correlating electron micrographs for both enzymes. The reaction of the red pigment product with thiocarbohydrazide is believed to establish a mercury-sulfur linkage which is apparently responsible for the pigment's surviving ethanol dehydration and epoxy embedding. Osmication of the tissue before dehydration appears to increase electron density of the product apparently by either chelation to the pigment product, by reduction of osmium tetroxide or by its decomposition with mercuric sulfide. The two acid hydrolases have been demonstrated within the cisternae of the Golgi and the endoplasmic reticulum and within the nuclear envelope of parenchymal cells of mouse and rat liver. Intracytoplasmic lipid inclusions in the livers of both C57 and C3H starved mice have shown a deposition of reaction product for acid phosphatase, whereas only C57 starved mice have shown a deposition of reaction product for β-glucuronidase. Lipid droplets in the rat preputial gland were sites high for β-glucuronidase, as has previously been demonstrated by two dissimilar techniques. Particularly helpful in evaluating the results of electron microscopy has been the use of physiologic controls as, for example, by comparison of findings made with the "high" tissue β-glucuronidase strain, C57, versus those of the "low" tissue β-glucuronidase strain, C3H. Localization of electron-dense product in lysosomes was clear, but not all of this can be attributed unequivocally to enzyme-produced density. The limitations of the technique and the question of nonspecific uptake of diazotized mercuric aniline are discussed. The findings for both hydrolases support the concept of the dual localization of acid hydrolases in lysosomes and in endoplasmic reticulum.

Hardness of Maraglas as affected by solvent-monomer interaction.

Acetone and propene oxide were mixed with equal volumes of Maraglas epoxy embedding medium. Retention of these solvents in the resin after cure at 60 C was measured. The retention of propene oxide was greater than that of acetone and appeared to be due to a chemical interaction with the monomer. The reaction was catalysed by benzyldimethylamine. Although 3-4% of added acetone was retained, no evidence that this resulted from a chemical interaction was obtained. Vacuum evacuation of added solvents prior to cure did not materially affect their retention. Retention of both solvents resulted in softer cured resins.

Millipore filtration of conjugating bacterial cells, with epoxy embedding for electron microscopy.

Broth cultures of Escherichia coli, strains Hfr G6 and F− 464 grown separately, were mixed (2 ml of each) and the mixture filtered through a 0.45 μ pore size, 25 mm diameter, MF-Millipore membrane. The membrane was placed, cell side up, on a nutrient agar plate and incubated 15 min at 37 C. Processing in the customary manner to include fixation, staining and epoxy embedding for electron microscopy followed. The technique provides greater concentration of cells, allows less opportunity for separation of conjugating cells and is less time consuming than procedures involving concentration by centrifugation and enclosure in agar as prerequisities to resin embedding.

Direct epoxy embedding for vertical sectioning of cells grown as a monolayer on millipore filter.

Cells cultured as a monolayer on MF-Millpore GSWP. 0.22 μ pore size, filter were fixed, dehydrated, and examined by phase-contrast microscopy with the filter immersed in a 1:1 mixture of xylene and the embedding medium. The membrane was cut into 2 × 20 mm strips, and each strip which was selected for desired cells was embedded vertically in a BEEM capsule. Thus direct embedding which allowed edgewise sectioning of cells was obtained without removing them from the culturing support.

Epoxy embedding of thin-layer material in commercially available vinyl cups for light and electron microscopy.

Epoxy embedding of cell cultures, impression smears and settled cell suspensions is performed in small vinyl cups, in which the entire sequence of culture, fixation, dehydration and embedding is feasible. The cup is readily stripped from the polymerized block to allow selected areas to be marked, photomicrographed by light, and thin sections of the selected parts cut for electron microscopy. The vinyl cups were obtained from Fabri-Kal Corp., Kalamazo, Mich. 49001 in sheets of 66 cups, each cup measuring 15 mm in diameter and 10 mm deep.

Polyester sheeting (Melinex O), a tissue-culture support easily separable from epoxy resins after flat-face embedding.

Pieces of Melinex O polyester films (0.003 in. thick) can be used as substitute for glass in flat-face embedding in epoxy resins for electron microscopy. Melinex sheeting can be obtained from Imperial Chemical Industries. It can be sterilized by alcohol or by autoclaving. It is transparent and it is resistant to customary cytological processing fluids. It separates readily from the resin block wihout the use of water and without cooling.

Ultrastructure of Trypanosoma lewisi: Flagellum, Microtubules, and the Kinetoplast

SYNOPSIS. With the technique of preselecting pure suspensions of trypanosomes for subsequent electron microscopy and with epoxy resin embedding after glutaraldehyde fixation, we have re‐examined the ultrastructure of Trypanosoma lewisi with particular reference to the flagellum and pellicle. The peripheral flagellar tubules exhibit clockwise asymmetry; however, sub‐tubule B of the peripheral tubules is divided into a doublet by a diagonally running anti‐clockwise arm that originates from the median diaphragm. Projections which seem to be continuous with the counter‐clockwise arms extend into a “basket shaped” intra‐flagellar structure. This structure, originating distal to the flagellar pocket and running anteriorly within the flagellar membrane, may play a role in maintaining the rigidity of the flagellum.The central flagellar tubules have a double helical substructure and dense cross‐striae (diameter 50 Å), and are regularly arranged at distances of approximately 250 Å. The central tubules arise from separate kinetosomal plates that lie at two different elevations. One central flagellar tubule originates from a flattened kinetosomal plate which makes contact with the peripheral tubules. The other central tubule passes through the flattened plate to fuse with an underlying “disc‐like” plate.The primary kinetosome exhibits the uniform triplet pattern of tubules at its most proximal region. A pair of tubules closely associated with one of the triplets represents two of the four subpellicular tubules which penetrate the cytoplasm in the region of the flagellar pocket. Sub‐pellicular tubules of dimensions similar to the flagellar tubules form a uniform cytoskeleton in all regions of the cell except in the area of the flagellar pocket.A desmosome‐like structure maintains continual cell membrane contact with the flagellum and the pellicle. This structure is designated the attachment zone. The fine structure of the kinetoplast is demonstrated. The dense intra‐mitochondrial elements (presumably containing DNA) are actually tubules, embedded in a moderately dense matrix. Morphological observations indicate that this organelle is probably primarily concerned with the ontogenesis of mitochondria, and several mitochondria extend from it into the cytoplasm of the cell.

In Situ Embedding in Epoxy Resin of Tissue Cultured in Leighton Tubes; Selection of Single Cells for Electron Microscopy

Processing is carried out in the original culture vessel as follows: fixation in a 1.0% solution of phosphate-buffered OsO4 at 5 C, 30 min; dehydration in ethanol, 30 min; propylene oxide, 30 min; infiltration with a 1:1 mixture of resin and propylene oxide, 30 min; and undiluted resin at 5 C, 12 hr. This resin is replaced with 8 ml of fresh, and the tube is placed horizontally in an oven. Araldite is polymerized at 55 C for 21 hr; Maraglas, 60 C for 24–48 hr. Immediately upon removal from the oven, the hot tube is plunged into an ice-water bath at 0–0.5 C. The tube usually cracks extensively, and after 8–10 min soaking, the glass fragments can be easily removed. If a tube fails to break, it can be cracked mechanically, then allowed to soak until separation occurs. Advantages of the method are that an entire culture can be embedded, the resin cast separates cleanly, and any part of the culture becomes available for light and electron microscopy.