A Plastic Embedding Medium for Thin Sectioning in Light Microscopy

Tissue blocks with surface areas up to 2 cm² can be sectioned at 1 or 2 μ after embedding in a medium consisting of: methyl methacrylate, 27 ml; polyethylene glycol distearate MW 1540, 6 gm; dibutyl phthalate, 4 ml; and Plexiglas molding powder A-100, 9 gm (added last). The methacrylate mixture is polymerized at 50° C by benzoyl peroxide, 0.8 gm/ 100 ml of methacrylate. The polymerized matrix is transparent and the blocks can be cut on a rotary microtome with a steel knife. The plastic can be removed from sections with acetone prior to staining. Artifacts caused by embedding and sectioning are negligible     

Serial Sectioning of Insects with Hard Exoskeleton by Dissolution of the Exocuticle

The method reported here was designed to produce paraffin serial sections as thin as 5 Mm of insects or other arthropods with a hard cuticle. Heads and abdomens of Apis mellifera, Eristalomyia tenax and Tenebrio molitor were fixed with Schaffer's liquid, dehydrated with 80% ethanol, 90% ethanol, two changes of 100% isopropanol (2 hr each) and 12 hr in a 1:1 mixture of paraffin (58 C melting point) at 60 C. They were molded in paraffin after 12 hr of infiltration under a partial vacuum at 60 C. Large body openings of objects were sealed with paraffin to prevent infiltration of solvents.

Thereafter, the outer paraffin was removed manually and with xylene (15 min); the cuticle was rehydrated with 100% isopropanol and 100% ethanol (15 min each). The objects were then treated with Sputofluol (Merck; a mixture of NaOH and NaCIO) until they became white or their colorless endocuticle was stainable with aniline blue WS (C.I. 42755) after rinsing in a 50% acetic acid solution (v/v). They were then dehydrated with 100% ethanol and 100% isopropanol (15 min each) and subsequently re-embedded in paraffin. They were molded, sectioned, stained and mounted as usual.     

A New Embedding Medium for Firm Materials Applied to the Sectioning of Plastic Vascular Prostheses

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.     

Methacrylate Embedding and Sectioning of Calcified Bone

Twenty-five milliliter aliquots of ethyl-butyl (1:1) methacrylate were polymerized at 6 or 7 initiator concentrations using 3 polymerization temperatures, both in air and in a water bath. Duplicate series were polymerized with and without vibration, pre-polymerization, and exclusion of oxygen. Hardening times and maximum temperatures reached within the samples were recorded. Vibration and the exclusion of oxygen had no effect. Prepolymerization, increasing polymerization temperature and increasing initiator concentration all decreased the hardening time and increased the maximum temperature. Polymerizing in a water bath rather than in air reduced the maximum temperature by 25-40°C and lengthened the hardening time about 1 hr. An initiator concentration of 0.4% Luperco CDB in ethyl-butyl methacrylate and a water-bath temperature of 45°C were selected for tissue embedding. The hardening time was 8 hr and the maximum temperature during polymerization was about 60°C.

Split rat femora and tibiae were freeze-dried and vacuum-infiltrated with acetone, absolute alcohol or monomer. The acetone or alcohol-fixed specimens were subsequently infiltrated with monomer. The specimens were transferred to 1 oz bottles, prepolymerized syrup added, and polymerized. No consistent differences between specimens treated by these methods were noted. Five-micron serial sections could be cut using a Leitz sledge microtome with a modified knife if the block was coated with paraffin between sections.     

Embedding and Sectioning Undecalcified Bone, and its Application to Radioautography

A method is described for embedding and sectioning hard, undecalcified bone, which is designed for use by technical personnel. Bone fixed in a variety of ways is progressed through alcohols to ether-alcohol and then infiltrated with ether-alcohol solvented plastic (plasticized nitrocellulose) by a combination of centrifugation and high pressure embedding technics. The ether-alcohol is evaporated in a partially closed container in a manner similar to that employed in celloidin embedding, but differs from the latter by the removal of all of the solvent. Celloidin is the source of nitrocellulose and Amoil-S, the added plasticizer. Undecalcified adult bone of all types is readily cut at a thickness of 5-8μ on a heavy duty sliding microtome. The sections are then mounted on gelatinized slides. The procedures for preparing strip film radio-autograms of bone sections and subsequent staining of the preparation are given. The results obtained are illustrated.     

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.     

Carbowax for Embedding and Serial Sectioning of Botanical Material

Plant material infiltrated with gradually increasing concentrations of Carbowax 400, followed by Carbowax 1540 and finally a 19:1 embedding mixture of Carbowax 1540 and 4000 showed minimum shrinkage. Quantitative measurements of shrinkage in tissue of potato tubers gave the following amounts: fixation and washing, about 4%; transfer from water directly to 70% Carbowax 400, 5176; from water through a graded series (5, 10, 15, 20, 30, 40, 50 and 60% Carbowax) to 70%, only 2.5% shrinkage; with an additional 1.5% occurring in transition to the embedding mixture. Dry ribbons are placed on adhesive-coated (gelatin, 5 gm; water, 120 ml; glycerol, 40 ml; phenol, 2 gm) slides in a humidity chamber. In 10-15 min enough moisture is absorbed by the ribbon to float the sections out gently and bring them in contact with the adhesive. Slides are then dried 5-10 min at room temperature. To remove minor wrinkles, the sections are subsequently flooded with water, then redried 12-24 hr; after which, they are ready for staining.     

Orienting and Embedding Small Objects in Paraffin

A beaker filled with ice cubes and water is covered with a plate of sheet metal. Over this is placed a paper embedding boat full of molten paraffin, and an inch or two above it is held a 60-watt soldering iron. The height of the soldering iron is then adjusted so that the paraffin at the lower half of the embedding dish becomes semisolid and the upper half stays liquid. By means of warmed forceps, the specimen is transferred to the dish, where it sinks to the top of the semisolid layer. Here, it is oriented by means of a warmed dissecting needle in relation to a pencil mark previously made on one side of the paper boat. One may take as long as he needs to orient the specimen, and several specimens can be oriented one after the other in the same dish.     

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.     

Sandwich Embedding of Monolayers of Cells in Epoxy Resin for Ultrathin Sectioning

In this procedure for embedding monolayers of cells, the usual glass slides are replaced by plates of resin 1-1.5 mm thick. Unlike the open-face embedding technique, the present procedure uses only a few drops of unpolymerized resin, which are applied to the fixed and dehydrated cells. During polymerization this small amount of liquid resin spreads across a relative large area, leaves the cells covered by a very thin layer, and permits phase contrast observations through it. Ultrathin sections of a particular cell encircled by a rotary scriber can be obtained by sectioning the resin slide, which has been trimmed and mounted directly in the specimen holder of the ultramicrotome.     

Embedding of Neural Tissue in Agarose or Glyoxyl Agarose for Vibratome Sectioning

Agarose was used to embed the brain or spinal cord of lampreys or rats before cutting vibratome sections. Agarose embedding was compatible with immunocytochemistry or the use of horseradish peroxidase as a neuroanatomical tracer. Concentrated agarose with high intrinsic gel strength was optimal for embedding glutaraldehyde fixed neural tissue. A quick procedure was to blot tissue and embed in 5% (w/v] Sigma type I-A or Litex type LSL agarose at 45-55 C dissolved in 50 mM neutral-pH TFUS buffer before cutting 50-100 μm vibratome sections. An alternative procedure that improved retention of tissue sections in the agarose was to rinse the tissue in H₂0, blot and embed in 5% (w/v] Sigma type I-A or Litex type LSL agarose at 45-55 C dissolved in H₂0, then equilibrate the block overnight in buffer. Phosphate buffer prevented complete dissolving of agarose. Tissue could be covalently linked to the embedding matrix using a novel aldehyde-derived agarose (NuFix® FMC BioProducts). Slices of spinal cord from neonatal rats could be cut after embedding in 5% FMC Seaprep® agarose in rat Ringer's at 23-26 C.     

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.     

The Use of Ethylenediamine in Softening Hard Plant Structures for Paraffin Sectioning

Ethylenediamine has been used as an agent for softening very hard woods prior to sectioning on a sliding microtome. The use of ethylenediamine is recommended for two additional uses: for preparing 1) soft woods in which wide, thin-walled tracheids or vessels tend to collapse during sliding microtome sectioning and 2) plant tissues with sclerenchyma mixed with soft-walled cells (bark, leaves, fruits, etc.) which frequently fail to section well. After softening in ethylenediamine, material is washed, infiltrated, and embedded in paraffin. Preliminary sections are made with a rotary microtome, just exposing the cut surface of the material; this exposed surface is soaked overnight in water. Sectioning is then continued. Sections produced in this fashion are considerably improved. The wood and pith of Podocarpus ustus, a parasitic conifer from New Caledonia, is used as an object to demonstrate improvements in sectioning by the ethylenediamine-paraffin method. Thinner sections with minimal tearing, cell collapse, and unevenness are produced. Sections can be handled easily and stained more effectively than unmounted sections. Variations in timing and in treatment are recommended to suit different materials. Ethylenediamine, used with reasonable caution, is much less hazardous than hydrofluoric acid and is more effective in softening plant material. The ethylenediamine method may be used routinely on any material difficult to section because of hardness.     

Serial Sectioning of Tissues in Glycol Methacrylate by Supplementary Embedding in a Paraffin-Plastic Matrix

Glycol methacrylate, while offering certain advantages over paraffin as an embedding medium, is difficult to use because it will not ribbon. Rohde (1965) developed a method for producing ribbons of methacrylate sections, but we had little success with it because the ribbons tended to fall apart when even slight stresses were applied to them. We have therefore made use of the principle of double embedding, as this has been used for obtaining serial sections of material embedded in nitrocellulose.     

Pollinators and Other Flying Insects inside and outside the Fukushima Evacuation Zone

Following the accident at the Fukushima Daiichi nuclear power plants in 2011, a large evacuation zone was imposed in an area where residents had historically managed forests and farmlands. Thus, the human activities that had maintained biodiversity and ecosystem services in the zone were discontinued. Such change can affect insects, a biodiversity component that is relatively tolerant to radiation exposure. In this study, we investigated flying insects, including pollinators, important ecosystem providers inside and outside the zone, using Malaise traps. The results showed that the number of individuals of Xylocopa appendiculata, the largest Apidae species in the region, was significantly lower inside the evacuation zone than outside it, whereas those of other insects were not lower significantly. Although we suggest that flying insects and their ecosystem services (i.e., benefits from them such as pollination) 3 years after the disaster were not critically impacted, it is important to monitor the long-term effects of the evacuation in the future.     

A Simple Method for Paraffin and Plastic Embedding of Protozoa

SUMMARY. To avoid multiple centrifugation and considerable losses of material in preparing protozoa for paraffin and plastic embedding a simple method has been worked out, requiring only two centrifugations, one before and one after fixation. During the second centrifugation, which is done at 700 g for 10 minutes, the organisms form a cohesive pellet which may be removed from the centrifuge tube by means of a fine spatula and handled as a piece of tissue through the whole process of dehydration and embedding.     

Interruptions in Single Strands of the DNA in Slime Mold and Other Organisms

The molecular weight of single-stranded DNA from the slime mold Physarum polycephalum has been determined by alkaline gradient centrifugation. The average molecular weight during DNA synthesis (~1.5 × 10⁷ D) is less than that observed in nonsynthetic periods (~4 × 10⁷ D). On the basis of a chromosome number of 50 per nucleus and a DNA content of 1 μμg per nucleus, we are led to conclude that at pH 12 each chromosome dissociates into 300 (single-stranded) pieces of DNA. We have also compared the sedimentation profiles of single-stranded DNA from Escherichia coli, PPLO, and T2 bacteriophage. These data support the conjecture that each bacterial chromosome can be dissociated into 10 or 12 single-stranded pieces of DNA. Dissociation of DNA into multiple pieces under our experimental conditions is best interpreted in terms of interruptions in the continuity of the DNA either by naturally occurring gaps or at alkali-labile bonds.     

Development of a Plastic Embedding Method for Large-Volume and Fluorescent-Protein-Expressing Tissues

Fluorescent proteins serve as important biomarkers for visualizing both subcellular organelles in living cells and structural and functional details in large-volume tissues or organs. However, current techniques for plastic embedding are limited in their ability to preserve fluorescence while remaining suitable for micro-optical sectioning tomography of large-volume samples. In this study, we quantitatively evaluated the fluorescence preservation and penetration time of several commonly used resins in a Thy1-eYFP-H transgenic whole mouse brain, including glycol methacrylate (GMA), LR White, hydroxypropyl methacrylate (HPMA) and Unicryl. We found that HMPA embedding doubled the eYFP fluorescence intensity but required long durations of incubation for whole brain penetration. GMA, Unicryl and LR White each penetrated the brain rapidly but also led to variable quenching of eYFP fluorescence. Among the fast-penetrating resins, GMA preserved fluorescence better than LR White and Unicryl. We found that we could optimize the GMA formulation by reducing the polymerization temperature, removing 4-methoxyphenol and adjusting the pH of the resin solution to be alkaline. By optimizing the GMA formulation, we increased percentage of eYFP fluorescence preservation in GMA-embedded brains nearly two-fold. These results suggest that modified GMA is suitable for embedding large-volume tissues such as whole mouse brain and provide a novel approach for visualizing brain-wide networks.     

The Orientation of Rhodopsin and Other Pigments in Dry Films

When rhodopsin in a gelatin film is dried, the rhodopsin chromophores orient primarily in the plane of the film. When the film is wetted, the chromophores disorient. These changes are reversible. When rhodopsin in a wet film. is bleached in the presence of hydroxylamine and redried, the retinal oxime which results is oriented more perpendicularly to the plane of the film. These orientations in dry gelatin films resemble those in the disc membranes of rod outer segments. A variety of other proteins are similarly oriented in dry gelatin films: methemoglobin, cytochrome c, phycocyanin. Films of methemoglobin and cytochrome c display prominently the high Soret band near 410 nm when measured with unpolarized light passing through the face of the fim, but display no Soret band at all with light passing through the edge of the film. All of these orientations imply a large asymmetry of the protein micelles, perhaps conferred upon them by linear polymerization in the course of drying. Such asymmetry can be demonstrated directly with rhodopsin. A wet paste of rhodopsin-digitonin micelles, sheared between glass slides, becomes highly oriented, the rhodopsin chromophores lining up in the direction of shear, the retinal oxime produced by bleaching orienting more perpendicularly to the shear.