A Simple Device for Holding Plastic Embedded Specimens During Dissection

Work in our laboratory involves the ultrastructural analysis of the organ of Corti in the inner ear (Hunter-Duvar and Mount 1978). Due to its fragile nature, dissection of the organ is difficult without causing mechanical damage. To overcome this problem the entire inner ear is embedded in plastic and polymerized prior to dissection of the organ of Corti (Bohne 1972, Spoendlin and Brun 1974). A method was sought to hold the embedded specimen securely which would allow adjustments in orientation during dissection.     

A Rapid Plastic Embedding Technique for Preparation of Three-Micron Thick Sections of Decalcified Hard Tissue

A 24 hour start-to-finish method is described for the preparation of three-micronthick sections of decalcified hard tissues. Following acetone dehydration, the tissue to be embedded is infiltrated under vacuum with a series of graded clearing solutions which approach the content of the final methyl methacrylate mixture. After overnight in a 35 C oven, the plastic is polymerizd by four hours heating at 42 C. Three-micron-thick sections are then easily prepared using a Jung microtome for high resolution histologic or detailed autoradiographic procedures.     

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.     

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.     

Simple and Durable Staining of Thick Sections of the Human Brain for Macroscopic Study

Observations on gross morphology of the human brain is greatly facilitated by enhancing the contrast between gray and white matter. The proposed technique is much more simple than the generally recommended Mulligan method and its variations. Moreover, there is no loss of stain since the fugitive surface impregnation, obtained by the Mulligan method, is replaced by a thoroughgoing block-staining procedure with the nonfading copper phthalocyanine dye astra blue. Staining procedure: wash formalin-fixed brain slices overnight in running tap water. Place slices in performic acid for 1 hour. Wash in running tap water. Place slices individually in staining solution consisting of 0.1 g astra blue (Merck) in 1000 cc distilled water and 1 cc HCl (37%), for 12-24 hours. Wash in running tap water. Embed in gelatin and mount in plastic cuvettes.     

A Method for Making Ribbons of Semithin Plastic Sections

Epoxy resin sections form strong, heat resistant ribbons if, prior to sectioning, contact cement has been painted onto the leading and trailing faces of the block. The forming ribbon floats onto a drop of water held in place by a wax line drawn across the back of the glass knife parallel to the cutting edge. A long trough made from stainless steel tubing is inserted horizontally into the drop, and as the ribbon lengthens it is directed into the trough. The ribbon can be carried in the trough to a hot plate for expansion and then poured onto a slide for mounting. The serial ribbons obtainable by this simple procedure greatly facilitate three dimensional reconstruction of fine tissue structures.'     

A Simple Procedure to Obtain One-Micrometer Sections of Routinely Embedded Paraffin Material

By freezing blocks of paraffin-embedded tissues to a convenient temperature it is possible to obtain routinely 1 µm sections that can be further processed as normal thicker sections. Normal and disposable steel knives can be used and the staining time should be increased in most procedures. Gradual freezing of blocks to the temperature of dry ice is the simplest and safest way to obtain an adequate temperature. The best results were obtained using as fixative 4% paraformaldehyde in phosphate buffered saline solution.     

Macrophotography of Thick Sections and Gels with A Regular Photographic Enlarger

At present, we have a program at Yerkes Regional Primate Research Center for the gross mapping of histochemical localization of various enzymes in the squirrel monkey brain and spinal cord, to characterize various regions. For these studies, sections about 50 μ thick must be used. The preparations measure from 4 × 5 mm in transverse sections of spinal cord to 20 × 40 mm in sagittal sections of cerebral hemispheres. Because of their dimensions, an ordinary camera attached to a microscope or even a plate camera could not be used satisfactorily for photographing them. However, projection with a darkroom enlarger has given very satisfactory pictures.     

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.     

Wrinkle-Free Plastic Sections for Light Microscopy

Plastic sections 0.5 to 2 μm thick are routinely used for light microscopy. Although plastic sections have several advantages over paraffin or celloidin sections, a problem that is often encountered with plastic sections is wrinkling (Fig. 1). Wrinkling occurs during staining when sections dried on glass slides are covered with stain and heated to hasten the penetration of the stain. Mounted sections heated on glass slides, but not stained, ordinarily lack wrinkles, even when examined with phase contrast optics. Similarly, mounted sections covered with stain, but not heated, lack wrinkles; unfortunately, such sections fail to stain adequately. Unmounted sections floated on heated drops of stain also lack wrinkles (Millonig 1980). Thus, it is clear that wrinkling occurs only when mounted sections are covered with stain and heated.     

A Water Control Device for Mounting Serial Ultrathin Sections

It is important to know the tissue reactions taking place in or near the wall and surroundings of plastic vascular prostheses transplanted into an organism. As is known, the porosity of the vascular prosthesis plays a significant role in the morphogenesis of vascular neogenesis (Sauvage 1971). for this purpose sections are needed in which the structure of the vascular prosthesis and the surrounding tissue are both well preserved.     

A Method of Affixing Separate Thick Sections from Materials Embedded in Paraffin

Botanical studies often require thick histological sections (for embryology, pollen and spore arrangement in tetrads, etc.). Study of the original position of the generative cell in Angiosperms, for example (Huynh 1972), requires paraffin sections bearing entire pollen grains with a diameter of up to 80 μm. However, it is impossible to obtain ribbons with sections of such thickness. If the sections are affixed separately, they do not hold so strongly to slides as do those mounted as ribbons; this difficulty increases with thickness of section. in addition, affixing sections separately with the required order and spacing is tedious and difficult, demands a great deal of time, and even so, is not always successful. the simple method described here can remedy such inconveniences.     

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 Methylene Blue-Azure Ii-Basic Fuchsin Stain for Epoxy-Embedded Tissue Sections

One micron-thick sections of tissues fixed in glutaraldehyde, or in glutaraldehyde followed by osmium tetroxide, and embedded in a variety of plastic resins were stained in a methylene blue-azure II solution at 65 C, then counterstained in 0.05% basic fuchsin in 2.5% ethanol at room temperature (24 C). Considerable variation was found in methylene blue-azure II staining times for different embedding media. Aged Epon-812 required less staining time than freshly polymerized blocks of Epon-812. The procedure is a simple, rapid staining technique suitable for photomicrography and tissue orientation for electron microscopy.