Sectioning of Undecalcified Bone; with Special Reference to Radioautographic Applications

A rapid technic for the preparation of 6 μ serial sections of undecalcified bone is described. The specimen is fixed and dehydrated in acetone and ether. It is then treated with a 1:1 mixture of the monomers of ethyl and n-butyl methacrylate catalyzed with benzoyl peroxide. The monomers are removed with ether and the ether is removed under vacuum. Infiltration is accomplished under vacuum using a partial polymer of the same mixture of monomers. Polymerization is completed in 36 hours under pressure at 50°C. The tissue is sectioned with a heavy-duty microtome, the sections are mounted on glass slides and nuclear emulsions applied. Young and adult bone of laboratory animals and man have been cut successfully. Microscopic structural detail is preserved and there is no evidence of translocation of the radioactivity.     

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.     

Block Staining with Hematoxylin, Gelatin Embedding and Serial Sectioning of Decalcified Bone

Decalcified pieces of bone can be stained prior to sectioning by washing and placing in undiluted Harris' hematoxylin (potassium alum). This stains the borders and contents of Haversian canals, lacunar margins, osteocyte nuclei, cementing lines and canaliculi. Embedding in 15% gelatin gives a flexibility which allows mounting of large numbers of serial sections with a minimum of tearing and folding. Frozen sections from the microtome knife are placed in water-filled depressions of chemists' porcelain spot plates, or other containers in the case of large sections. Long pieces of bone can be cut into smaller blocks, which are placed in additional gelatin. This allows complete sectioning of the block and examination of structures continuing throughout the bone.     

Staining of fresh, Undecalcified, thin Bone Sections

Fresh, undecalcified bone sections can be reproducibly and reliably stained by any of the following procedures: (A) Basic fuchsin, 1% in 30% alcohol, 48 hr, 22°C. (B) AgNO₃, 0.033 M, 48 hr, 22°C; washing 48 hr in a large volume of distilled water; exposure to light to develop the color. (C) Metallic sulfides (Co⁺⁺, Pb⁺⁺, Hg⁺⁺, Cu⁺⁺): the nitrate of the metal, 0.033 M, 48 hr, 22°C; then Na₂S, 0.033 M, 48 hr, 22° C. (D) Alizarin Red S, 0.1% solution in distilled water, 48 hr, 22°C; differentiated 48 hr at 22°C in weakly alkaline water, pH about 8. (E) KMnO₄: boiling 8-10 min in a 0.1 N, solution. With the exception of D the surface stain must be ground off the section for microscopic examination of its interior. Stain concentration, time and temperature can be altered to suit specific needs.     

A Technique for Embedding Undecalcified Bone Samples for Detecting Alpha-Emitters Using Vacuum Impregnation with Spurr's Resin

A method has been developed by which large samples of mineralized bone, containing an alpha-emitter, can be embedded in Spurr's resin in a fraction of the time required by conventional methods. Bone samples were freeze-dried or fixed and dried prior to impregnation with Spurr's resin under vacuum. Sections were cut for the preparation of either alpha-track or fission-track autoradiographs using the solid state detector CR-39. This method is applicable to samples containing a mobile form of a radionuclide that may be translocated during the processes of fixation and dehydration of the specimen.     

Undecalcified Bone Preparation for Histology,Histomorphometry and Fluorochrome Analysis

Undecalcified bone histology demonstrates the micro-architecture of bone. It shows both the mineralised and cellular components of bone, which provides vital information on bone turnover or bone formation and resorption. This has tremendous importance in a variety of clinical and research applications. It yields beautiful images¹ and allows for techniques such as fluorochrome assessment and histomorphometry². Fluorochrome analysis is a technique where fluorescent dyes that bind to calcium are injected at a particular time point, which allows for quantification of the amount of mineralisation at that given time. Histomorphometry is a process of bone quantification at the microscopic level. Performing undecalcified bone histology is technically challenging, particularly with large size specimens. It requires variations in technique from those used in standard paraffin embedded histology. This video illustrates the process of producing good quality sections and demonstrates the technical difficulties and methods with which to overcome them. Specimen preparation, fixation and processing are achieved with a manner similar to other soft tissues, however due to the density and lower permeability of bone considerably longer fixation and processing times are required, often taking several weeks. Embedding is achieved using a supporting medium with similar or equal hardness and density to the bone such as methacrylate- based resins, but unlike paraffin infiltration and embedding, this is an irreversible step. Sectioning can be achieved by grinding which produces a thicker section, which is optimal for studies such as fluorochrome analysis. This is best achieved using a diamond blade on a macrotome. Alternatively, thinner sections can be produced for light microscopy and this is achieved using a sledge microtome with a very sharp blade. The sledge microtome provides the additional strength and stability required for large, hard blocks. Resin embedded sections can be stained with a variety of stains, which are demonstrated.Download video file.^{(88M, mp4)}     

Epoxy Resin Embedding in Contrast Radioautography of Bones and Teeth

Contrast, or low-resolution, radioautographs of teeth and large specimens of bone are made from plane, polished surfaces of such material embedded in plastic. The specimen is cut with a motor-driven circular saw, fixed, dehydrated, and defatted with anhydrous acetone and ether. It is then infiltrated with a mixture of epoxy resin and reactive hardener. The resin-infiltrated specimen is placed in an easily constructed mold of aluminum and Lucite. Polymerization is complete in 4-6 hr. The surface of the specimen is exposed by machining and/or abrading. The prepared surface is apposed to an X-ray type emulsion to produce a radioautograph. The resolution obtainable is estimated to be 100-200 β.     

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.     

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.     

Diethylene Glycol Distearate Embedding and Ultramicrotome Sectioning for Light Microscopy

Diethylene glycol distearate can be used as an embedding medium for light microscopy. Two infiltration changes of about 6 hr each in the melted wax (melting point 47-52 C) are required before the final embedding which is done in 00 gelatin capsules for sectioning in the ultramicrotome by the procedure used in electron microscopy. Serial sections 1-2 μ thick can be cut without difficulty. No cooling devices are necessary for trimming and sectioning at laboratory temperature. Sections rarely become detached from the slides. The staining characteristics of the tissues are the same as when embedded in paraffin. For fluorescence microscopy, essentially the same procedure is followed. Tissues are not distorted and the intracellular structures are well preserved.     

Polyester Resin Embedding for Sectioning of Hard and Soft Tissues

Soft and calcareous tissues embedded in polyester resin may be cut on a sledge microtome to produce thin sections of 3-4 β thickness. Fixed tissues, dehydrated in ethyl alcohol, cleared in methyl benzoate and chloroform, are taken into a wide-necked bottle containing equal parts of polyester resin and chloroform with 0.75% catalyst. The bottle kept in water bath at 37°C is connected to a vacuum pump. With the evaporation of the chloroform under reduced pressure (approximately 10 mm Hg) infiltration is complete. Tissues transferred into a blocking form containing pure polyester resin with 1.5% catalyst are polymerized at 37° C until blocks are firm (48 hr or more). Blocks are prepared with at least 5 mm margin of plastic surrounding the tissue. The edge of the block adjacent to the knife is then filed at an angle of 45° to the cutting movement. Sections are cut with a wide-backed biplanar knife having a cutting edge of 40-44° positioned at an angle of 30° to the plastic block. As the resin is permeable to most stains, staining is carried out through the plastic Sections carried through staining procedures in wire baskets are floated onto slides and mounted in polystyrene; the cover-glass is compressed with a spring-clamp. Microscopic examination shows no staining of plastic, minimal shrinkage and good cellular detail.     

A Thionin Stain for Visualizing Bone Cells, Mineralizing Fronts and Cement Lines in Undecalcified Bone Sections

A staining method is described using thionin, for undecalcified deacrylated bone sections. RNA is stained purplish violet, allowing still active osteoblasts to be distinguished from lining cells. Staining intensity of mineralized bone is related to the degree of mineralization. Mineralizing fronts and cement lines are visualized clearly. Lamellae show an alternate pattern. Histomorphometric parameters such as osteon thickness and interstitial bone thickness can be measured without using polarized light. The mineralizing front can be assessed and expressed as a percentage of the osteoblast-covered interface between osteoid and mineralized bone. The stain is also useful for qualitative assessment of metabolic bone disease. Thionin stained sections can be kept for at least one year when stored hi the dark at 7 C.     

Oriented Embedding of Biological Materials and Accurate Localization for Ultrathin Sectioning

Gelatin capsules with rounded ends clipped off and open ends moistened, affixed to a glass slide and sealed with a 15% gelatin solution are used to embed blocks of tissue in plastic. The surface of the slide serves as an orientation plane for structures of the tissue. The plane end of capsules of polymerized plastic containing no tissue is used in embedding frozen tissue sections. The plastic-infiltrated section is flattened against the capsule end under the weight of a 3/4 inch square of plate glass so that larger sections may be cut and surveyed. Embedding cultured cell monolayers grown on coverslips is accomplished in a comparable manner, but the square of plate glass is not needed as a weight. Block-face localization methods depend on the type of material embedded. With blocks of tissue it is achieved by moistening the face with xylene to develop relief. Thin tissue sections are examined by transmitted light, while cell monolayers are stained on the capsule end with methylene blue.     

Staining and Histochemistry of Undecalcified Bone Embedded in a Water-Miscible Plastic

Undecalcified bone fixed in a variety of fixatives and embedded in a new formulation of 2-hydroxy propyl methacrylate at 4 C has been sectioned at 1 to 5 microns. The embedding mixture contains 2-butoxyethanol as plasticizer and triethyleneglycol dimethacrylate as cross-linker. The accelerator was benzoyl peroxide and the catalyst was N,N-dimethylaniline. With proper embedding and care in sectioning it is possible to obtain sections with relatively little bone compression, excellent preservation of cellular elements, and a minimum of wrinkling. A wide variety of stains have been used for these sections and those reported here are Gill's hematoxylin-eosin, Nocht's azure-eosin, Feulgen, Hoechst 33258 (bisbenzimid H 33258), methyl green-pyronin, PAS, alizarin red, and von Kossa silver stain. There was excellent preservation of acid and alkaline phosphatase activities. A new method of prestaining immunofluorescent labeling was also applied to bone and examples of staining with anticollagen I and antifibronectin are presented.     

Plastic Embedding, Orientation in the Mold and Tape-Supported Sectioning of Sclerotized Insects and Other Hard Objects

Sections of large specimens such as whole honeybees or beetle adults embedded in plastic usually are difficult to cut with a constant thickness. The sections also compress and roll. Sections of even thickness have been obtained by using a mixture of methacrylates (ethyl, 1:butyl, 3) and by firmly supporting the block in the microtome with a special holder. Scotch tape #810 applied to the block before each section is cut eliminates section compression and rolling. The sections are attached to slides with 2% celloidin in an absolute alcohol-methyl benzoate mixture (5:5-7:3); and the tape is removed with heptane. Large sections can also be cut from blocks of styrene mixed with butyl methacrylate. The specimens are oriented in the monomer in gelatin capsules by directing them into the desired plane among the fibers of a wad of absorbent cotton previously placed in the bottom of the capsule. The cotton is sectioned with the specimen but its fibers do not interfere, and remain outside the tissue.     

A Procedure for Preparing Undecalcified and Unembedded Bone Sections for Light Microscopy

We have developed a procedure for light microscopic investigation of undecalcified and unembeddedbone sections. Biopsy samples of human metatarsus and femur and rat femur were fixed in aldehydes and sectioned with a cutting machine equipped with a diamond saw blade. Free sections 100-150 μm thick, stained with toluidine blue and von Kossa, did not show artifacts following the cutting, and the spatial relations of mineralized and nonmineralized components remained intact. Compact and trabecular bone, bone marrow and all cell types appeared well preserved and easily recognizable. Our procedure provides a simple and rapid method for preparing bone sections which undergo no chemical treatment other than fixation. This method is a useful alternative to standard histological protocols for studying bone specimens.     

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.     

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.