Glass Knife Adapter for Cutting Epoxy Sections on a Conventional Rotary Microtome

Electron microscopy-style fixation followed by epoxy plastic embedding is often now the method of choice for preparing tissue even for light microscopy; I have found it excellent for fluorescence, autoradiographic and conventional histology (Shaw 1972, 1977). Sections more than about five microns thick can be cut on a really sharp steel knife if the plastic is reasonably soft (Stretton and Kravitz 1973, Shaw 1972), but this is much easier and knife marks are reduced if extra-wide glass knives are used on a special-purpose intermediate microtome like the Sorvall JB-4. Recent budgetary restrictions made us defer purchase of such a microtome, and some alternative had to be devised. I report here a simple but rugged adapter for glass knives which replaces the steel knife in a conventional Leitz rotary microtome and allows thin plastic sections to be cut as easily as with a more sophisticated cutter. It could be adapted for any rotary microtome, and can be readily constructed in most machine shops for negligible cost.     

A Method for Breaking Glass Knives Slowly

There is a general belief among electron microscopists that the speed of the break in forming the edge of a glass knife is of great importance in determining its quality. Slow breaks seem very desirable (Hayat 1970).     

Stirring During Freeze-Substitution

There is a general belief among electron microscopists that the speed of the break in forming the edge of a glass knife is of great importance in determining its quality. Slow breaks seem very desirable (Hayat 1970).     

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.     

Tape transfer sectioning of tissue microarrays introduces nonspecific immunohistochemical staining artifacts

Abstract

Tissue microarrays place tens to hundreds of formalin fixed, paraffin embedded tissue cores into a paraffin block in a systematic grid pattern that permits their simultaneous evaluation in a single section. The fragmented nature of the tissue cores often makes sectioning of tissue microarrays difficult so that the resulting disks of tissue lose their shape, fracture or fall out of the paraffin section altogether. We have evaluated an alternative sectioning protocol for stabilizing the tissue microarray surface by placing an adhesive tape “window” over the face of the paraffin block prior to sectioning. Once sectioned, the tape/sections are transferred directly onto coated microscope slides, thereby avoiding routine floating of sections on a water bath. After sectioning with either the tape transfer or standard protocols, slides were stained either using hematoxylin and eosin or immunohistochemistry using antibodies to S-100 protein and the tissue specific antigens, keratin (AE1/3) and the leukocyte common antigen CD45. We found that the tape method produced thicker sections that were darker and more densely packed with loss of tissue definition compared to sections prepared using water bath flotation. Quantitative image analysis of immunohistochemical staining demonstrated that the tape method produced a higher incidence of nonspecific staining, which raised the potential for false positive staining.     

A Ralph Knife Holder Assembly for Use with the Sorvall “Porter-Blum” MT-1 Ultramicrotome

The superiority of plastic embedding for the production of high quality sections for light microscopy is well known, but the use of conventional glass knives with a cutting edge of approximately 4 mm has severely restricted the size of specimens in the past. Ralph knives provide a much longer cutting edge and adapters are available for certain models of microtomes and ultramicrotomes. A modified knife holder for use with the Sorvall “Porter Blum” MT-2 microtome was described by Gorycki and Sohm (1979); however, this is not suitable for the MT-1 model. We have therefore designed and made an adapter which enables Ralph knives to be used with this instrument. The design allows approximately 18 mm of cutting edge to be used on each knife, allowing larger specimens to be sectioned than with a conventional glass knife and reducing the frequency with which the knife needs to be changed when working with smaller blocks.     

A Novel Polystyrene Dish for the Production of Anaerobiosis

S ummary : The design and construction of a polystyrene dish to be used with alkaline pyrogallol for the production of anaerobic conditions is described. Paper mats are used to carry the reducing system, and the use of cellulose adhesive tape to effect an airtight seal is recommended.     

Improved Paraffin Sectioning with Etched Microtome Knife Edges

Carbon steel microtome knives etched with 0.1 N HNO₃ for 2 min display a very sharp cutting edge. Over a period of 3 yr no damage to the steel has been detected. The effect on paraffin sectioning was observed by comparing acid-treated knives with nonetched but well-sharpened ones. Sections of whole eyes cut with an etched blade showed approximately 15% less compression of the parffin matrix than those sectioned with an untreated knife. Tissues selected from routine autopsy material presented approximately 9% reduction in compression. As a result, excellent ribbons of sections could be cut from 5-7 μ and floated on water at 42—46 C with a minimum of folds or distortion. Etching improved sectioning when knife edges having bevel angles in a range of 31-39° were used, and also when the bevel was decreased to 20°, but the 20° edge gave impractically short service.     

Vitreous Carbon: A New Material for Making Microtome Knives

The quality of sections obtained by microtomy depends to a large extent on the quality and characteristics of the microtome knife itself. Despite the need for improved microtomy techniques, there have been few significant developments since the introduction of glass and diamond knives in the 1950's. The manufacture of microtome knives from vitreous carbon provides new possibilities for developing both improved methods and improved equipment for specimen sectioning. Vitreous carbon has unique physical properties that lend themselves to the generation of precision cutting edges. Such an edge can be obtained either by breaking a piece of vitreous carbon or by using lapidary techniques. The resultant edge seems well adapted to both thick and thin sectioning. The introduction of vitreous carbon as a sectioning tool offers a significant alternative to metal, glass and diamond knives.     

Are natural threats superior threats?

Threat superiority effects describe the reaction time advantage for locating threatening objects in a visual search paradigm, compared to locating visually similar non-threatening objects. They are widely reported for threats of both natural (snakes and spiders) and man-made (guns and knives) origins. Across two experiments, the current study contrasts threat superiority effects for natural and man-made targets. When targets are not depicted held, snakes and spiders tended to exhibit larger threat superiority effects, and were searched for with additional caution, than were guns and knives. When snakes and spiders were depicted held and weapons wielded, systematic differences between the natural and man-made threats disappeared. This means the advantage for threats of natural origin observed when all targets were depicted not held may be attributable to differences in animation – snakes and spiders are alive and may strike at any time if in your vicinity, whereas a weapon can only inflict harm if wielded. From these data there is no evidence that evolved visual sensitivities to the basic shapes of venomous animals support faster detection and response times to these animals than can occur to targets such as guns and knives, whose shapes must be learned. The selection pressures that led to the evolution of such sensitivities (observable even in infancy) may therefore lie in protecting young children and babies from envenomation, before they even have the cognitive capacity to understand the dangers that snakes and spiders pose.     

The Vibratome-Ralph knife combination: a useful tool for immunohistochemistry

A modified immunohistochemical technique is described for the improved detection of antigens. The method involves the use of the Vibratome combined with Ralph knives, which are easily manufactured with an LKB 2078 Histoknifemaker. The sectioning procedure was used with the peroxidase-antiperoxidase method of Sternberger and his collaborators for demonstrating growth hormone release-inhibiting hormone (somatostatin) and noradrenaline N-methyltransferase in neurones in the central nervous system of the rat. The morphological preservation of the tissue was good and cytological details were easily seen, especially in the thin sections (5-10 microns thick).     

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.     

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.     

Some observations on glass-knife making

The yield of usable knife edge per knife (for thin sectioning) was markedly increased when glass knives were made at an included angle of 55 degrees rather than the customary 45 degrees. A large number of measurements of edge check marks made with a routine light scattering method as well as observations made on a smaller number of test sections with the electron microscope indicated the superiority of 55 degrees knives. Knives were made with both taped pliers and an LKB Knifemaker. Knives were graded by methods easily applied in any biological electron microscope laboratory. Depending on the mode of fracture, the yield of knives having more than 33% of their edges free of check marks was 30 to 100 times greater at 55 degrees than 45 degrees.     

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.     

Reversible embedment cytochemistry (REC): a versatile method for the ultrastructural analysis and affinity labeling of tissue sections

Reversible embedment cytochemistry (REC) is a new method for revealing cellular ultrastructure and for improving access of intracellular targets to macromolecular affinity labels. Fully polymerized polymethylmethacrylate was dissolved in dichloromethane and infiltrated into fixed tissue-culture cells and tissues. After evaporation of the solvent, samples were left in hard plastic. Samples were thus embedded without exposure to chemical polymerization reactions that might damage tissue ultrastructure or antigenicity. Glass or diamond knives fitted with water troughs were used to cut sections 30-1000 nm thick. Since polymethylmethacrylate is composed of linear polymers that are not covalently crosslinked, the plastic was easily extracted from the sections by immersion in solvent. Subsequently, various preparative methods, including negative staining, critical point-drying, and platinum-carbon rotary shadowing, were used to provide detailed images of well-preserved cell structure for conventional and high-voltage transmission electron microscopy. Fluorescein-conjugated affinity labels were used to obtain subcellular distributions of target molecules in semi-thick sections of cultured cells and tissues for light microscopy. Colloidal gold-labeled antibodies were used to localize microtubules in sections of cultured cells by electron microscopy. REC is a versatile method that should find wide application in many studies of cellular function.     

Some Observations on Glass-Knife Making

The yield of usable knife edge per Me (for thin sectioning) was markedly increased when glass knives were made at an included angle of 55° rather than the customary 45deg;. A large number of measurements of edge check marks made with a routine light scattering method as well as observations made on a smaller number of test sections with the electron microscope indicated the superiority of 55° knives. Knives were made with both taped pliers and an LKB Knifemaker. Knives were graded by methods easily applied in any biological electron microscope laboratory. Depending on the mode of fracture, the yield of knives having more than 33% of their edges free of check marks was 30 to 100 times greater at 55° than 45°.