A simple method for collecting and mounting ribboned serial sections of epoxy embedded specimens

A simple and rapid method of handling ribboned serial sections of epoxy embedded specimens is described. Ribbons are cut from a block having the leading and trailing sides coated with contact cement. A scoop made from polyethylene tubing is used to remove a ribbon of sections from the boat of a glass or diamond knife and to transfer it to a pool of water on a microscope slide. Many ribbons (comprising hundreds of sections) can be mounted on a single slide. This method requires the construction of only one simple, inexpensive tool, the polyethylene scoop, and otherwise utilizes only items commonly available in the laboratory.     

Ultrastructural detection of DNA-incorporated bromodeoxyuridine in resin embedded brain tissue

Summary We describe a protocol for the ultrastructural detection of DNA-incorporated bromodeoxyuridine (BUdR) in resin embedded tissue by means of post-embedding immunogold labeling. The paraventricular zone of rat embryos brains was dissected, fixed either in paraformaldehyde or glutaraldehyde, and embedded in LR White. BUdR gold labeling was only found when thin sections were pretreated with 4 N HCl. Other DNA denaturing agents, such as Na ethoxide, formamide, formic acid, heat or HCl at lower concentrations were ineffective. Very little difference in the degree of labeling was found depending on the fixation. This method can be applied to investigate the fine structure of replicating cells in other in vivo conditions, such as human tumors.     

Ultrastructural detection of DNA-incorporated bromodeoxyuridine in resin embedded brain tissue

We describe a protocol for the ultrastructural detection of DNA-incorporated bromodeoxyuridine (BUdR) in resin embedded tissue by means of post-embedding immunogold labeling. The paraventricular zone of rat embryos brains was dissected, fixed either in paraformaldehyde or glutaraldehyde, and embedded in LR White. BUdR gold labeling was only found when thin sections were pretreated with 4 N HCl. Other DNA denaturing agents, such as Na ethoxide, formamide, formic acid, heat or HCl at lower concentrations were ineffective. Very little difference in the degree of labeling was found depending on the fixation. This method can be applied to investigate the fine structure of replicating cells in other in vivo conditions, such as human tumors.     

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

Summary 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 2 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.     

AFM of biological material embedded in epoxy resin

We present a simple method to extract morphological details from the block face of epoxy embedded biopolymers by AFM. It is shown that topographical contrast and the identification of small structural details critically depend on the procedure of sample preparation before embedding (chemical fixation or high-pressure freezing and freeze-substitution) and on the hardness of the embedding epoxy resin. Ethanol treatment of the block face of the sample after microtomy elutes non-cross-linked polymer chains and makes the smallest details of the embedded biomaterial amenable to detection. AFM (height and phase contrast) examination of the block face of accordingly prepared cells of Caenorhabditis elegans provides data that are comparable to TEM.     

Staining properties of alizarin red S for growing bone in vitro

The pH-metric titration curve of alizarin red S (ARS) showed that ARS ionizes in three stages: firstly the -SO3Na group, secondly the beta-hydroxyl group, and finally on the alkaline side the alpha-hydroxyl group. The titration of ARS solution with calcium ions indicates that complex and chelate formation between ARS and calcium are distinct. The in vitro staining experiments were carried out at different pH levels. Completely ionized ARS had the best affinity for growing bone surfaces. The next most effective staining occurred just prior to neutralization. The least effective staining by ARS was found after the neutralization point had been reached. The presence of calcium made the dye solution inactive for staining the growing bone in vitro.     

The use of alizarin red S to detect and localize calcium in gametophyte cells of ferns

An aqueous solution of alizarin red S containing chloral hydrate both clears intact chlorophyllous gemma cells of Vittaria graminifolia and stains for protoplasmic calcium. Verification that the stain was protoplasmic rather than in the cell wall was shown by a positive reaction in extruded protoplasm. Similar staining was found in extruded protoplasm of Onoclea sensibilis spores. Differentiating gemma cells show localized protoplasmic accumulations of Ca2+ at sites where asymmetric cell divisions initiate the formation of rhizoids, antheridia or vegetative cells. The staining properties of the dye depend on careful control of pH and the addition of appropriate amounts of KCl to the mixture. Treatment of Onoclea spores and Vittaria gemmae with 100 mM EGTA for 30 min nearly abolishes staining of their extruded protoplasts and also of intact cells of gemmae. The use of alizarin red S with and without chloral hydrate demonstrates different pools of protoplasmic Ca2+. When Onoclea spores are ruptured to extrude the protoplasm, both dye mixtures stain a peripheral, granular protoplasmic component. However, the chloral hydrate-containing dye also reveals Ca2+ associated with small particulate protoplasmic components. Extruded protoplasm of gemma cells stains intensely with alizarin-chloral hydrate, but does not stain with alizarin lacking chloral hydrate.     

A simple staining method for vaginal smears using red ink.

In adult rhesus monkeys each phase of 37 menstrual cycles was accurately classified by cellular identification of vaginal smears stained with red ink. This technic was simple, quick, and inexpensive, and produced well-stained slides in a few seconds.     

Gold labeling with wheat germ agglutinin and RNAse on osmicated tissue embedded in epoxy resin

Tissue of an insect, Lucilia cuprina, fixed conventionally in buffered glutaraldehyde and osmium and embedded in epoxy resin (epon or epon/araldite), provided sections which could readily be labeled with RNAse/gold and wheat germ agglutinin (WGA)/gold. This method offers labeling of tissues with improved contrast and allows the retrospective application of RNAse and WGA labeling to conventionally prepared tissues, without recourse to oxidizing/etching agents.     

Light microscopic localization of labile calcium in hypertrophied chondrocytes of long bone with alizarin red S.

A method which localizes labile 5% ethylene glycol-bis-(beta-amino-ethyl ether)N-N'-tetraacetic acid-removable calcium in spherules within hypertrophied chondrocytes and in pericellular matrix using alizarin red S (ARS) is described. Fresh blocks of epiphyseal cartilage approximately 1 mm thick were immersed into 0.5-2% ARS solution containing 7% mounted on glass slides in 7% sucrose or in glycerol-gelatin. The stained tissue blocks were also dehydrated in acetone, cleared in xylene and mounted in Preservaslide. The ARS precipitated ionic calcium as red Ca-ARS salt which was birefringent in polarizing microscope, stable in water at pH 4-9 and in nonpolar organic solvent but soluble in polar solvents, especially in dimethyl sulfoxide. In contrast, ARS-stained insoluble calcium phosphate was stable even in dimethyl sulfoxide. Calcium in the hypertrophied chondrocytes, therefore, was thought to be present in a readily ionizable state instead of as insoluble calcium phosphate. Since addition of 7% sucrose retained as well as improved ARS localization of cellular calcium, the calcium was believed to be present in an osmotically sensitive, membrane-bound cytoplasmic compartment. The ARS-positive labile calcium in spherules which develop in the hypertrophied chondrocytes as well as in the pericellular matrix at the zone of provisional calcification suggested a preparatory stage in the process of cartilage calcification.     

Detecting microcalcifications in atherosclerotic plaques by a simple trichromic staining method for epoxy embedded carotid endarterectomies

Atherosclerotic plaques have a high probability of undergoing rapid progression to stenosis, becoming responsible of acute coronary syndrome or stroke. Microcalcifications may act as enhancers of atherosclerotic plaque vulnerability. Considering that calcifications with a diameter smalller than 10 µm in paraffin embedded tissue are rather difficult to detect, our aim was to analyze microcalcifications on semithin sections from epoxy resin embedded samples of carotid endarterectomies using an original trichromic stain (methylene blue-azur B - basic fuchsine - alizarin red). We have compared samples stained either with our method, methylene blue-azur B alone or with Von Kossa staining, and methylene blue-azur B -basic fuchsine alone or with Von Kossa staining.Our method resulted to be simple and fast (ca. 2 min), it gives a sharp general contrast for all structures and allows to easy identify collagen and elastin. In addition, gray-green colour associated to intracellular lipid droplets evidences foam cells, which are particularly abundant in endarterectomies samples. Mast cells and their metachromatic granules are also well recognized. Calcifications over 0,5 µm are clearly recognizable. In conclusion, microcalcifications are clearly distinguished from the extracellular matrix in spite of their reduced dimensions. Methylene blue-azur B-basic fuchsine-alizarin red method is easy to use, reproducible, and is particularly suitable for the identification of microcalcifications in the morphological analysis of atherosclerotic plaques.Key words: atherosclerosis, carotid endarterectomy, calcification, polychromatic stain, epoxy resin, transmission electron microscopy.     

A simple method using PCR for direct sequencing of genomic DNA from frozen tumor tissue embedded in optimal cutting temperature compound.

Described here is a three-day protocol that directly yields DNA sequence after isolating and PCR amplifying genomic DNA from a small sample of frozen nasopharyngeal carcinoma tissue embedded in optimal cutting temperature (OCT) compound. The method is consistently successful, reproducible and will facilitate the rapid analysis of DNA sequence from very small samples.     

A silver method for counterstaining plastic embedded tissue

This report presents a method which can be used for counterstaining semithin sections of plastic embedded tissue. The sections are treated with a solution of silver lactate, followed by physical development. During the silver lactate treatment, silver ions are bound by various tissue components as metallic silver or silver sulfide. During physical development catalytic reduction of silver ions to metallic silver takes place where silver has been bound in the tissue, enlarging the silver deposits to microscopically visible dimensions. The amplified silver deposits give high contrast staining in yellow, brown and black suitable for both color and monochrome photography. The localization of the silver deposits is highly specific and may reflect several independent chemical processes. Examples in several tissues are shown.