Preparation of Thin Undecalcified Bone Sections by Rapid Manual Method

Sections from 3 μ to over 100 μ thick of fresh, unfixed, unembedded, unde-calcified and undehydrated bone are made by grinding 1 to 2 mm slabs of the desired orientation on waterproof carborundum abrasive paper, grit No. 320, 360 or 400. The manner of controlling the section is the crux of the technique. The section is held by wrapping a fresh strip of sandpaper around a 3' × 1' slide and accomplishing the grinding on a used piece of paper. The abrasive points on the fresh paper effectively prevent the section from sliding off the slide. The specimen is kept wet with water during the entire procedure. Sections are then stained, and excess surface stain can be ground off in similar fashion. After washing in dilute detergent solution to remove adherent derbis, the section is air dried and mounted in any nonacidifying resinous media. The method is suitable for wood and for fruit pits also.     

Autoradiography of Water-Soluble Materials in Plant Tissues

Samples of tissue with water-soluble substances are lyophilized and doubly-infiltrated with parlodion and paraffin. Sections are mounted on an adhesive-coated cover slip with chloroform. The reverse side of the cover slip is coated successively with a thin layer of Harleco resin and a thick layer of Kodak “Opaque”. A corner of the cover slip is broken off as a marker. The cover slip is assembled with a covering glass slide on a nuclear track plate (Kodak NTB-2) with protective coating, the section being in contact with the emulsion, and held in place during radioactive exposure. Before development, the cover slip and plate are exposed briefly to light and then disassembled. Following development of the plate and removal of the layer of resin and opaque from the cover slip, staining of the section is optional. Lining up of the section with its autoradiograph can be accurate within 1-5μ.     

Autoradiography of Water-Soluble Materials in Plant Tissues

Samples of tissue with water-soluble substances are lyophilized and doubly-infiltrated with parlodion and paraffin. Sections are mounted on an adhesive-coated cover slip with chloroform. The reverse side of the cover slip is coated successively with a thin layer of Harleco resin and a thick layer of Kodak “Opaque”. A corner of the cover slip is broken off as a marker. The cover slip is assembled with a covering glass slide on a nuclear track plate (Kodak NTB-2) with protective coating, the section being in contact with the emulsion, and held in place during radioactive exposure. Before development, the cover slip and plate are exposed briefly to light and then disassembled. Following development of the plate and removal of the layer of resin and opaque from the cover slip, staining of the section is optional. Lining up of the section with its autoradiograph can be accurate within 1-5μ.     

A Method for the Preparation of Serial Thick Sections of Plastic-Embedded Plant Tissues

A procedure is described in which thick sections (2-10μ or more) of plastic-embedded plant tissues are mounted in serial order on slides for use in routine light microscopy. Sections are cut with a steel knife on a rotary microtome while the block and blade are bathed with 40% alcohol. The cut sections are placed, in order, in 50% alcohol in the small wells of modified plastic trays where they become flat, pliable and suitable for subsequent handling. Sections remain separate and in correct order in the trays while they are stained, washed, and prepared for final mounting on slides. Mounting involves a simple and rapid procedure of transferring the sections to a slide and heating first on a 70-75 C hot plate (to slowly evaporate the water around the section and to partially affix the section) and then on a 100 C hot plate. This second heating ensures adhesion when xylene-base mounting media, which tend to loosen weakly adhered plastic from the slides, are used. The technique of staining the sections loose provides the following advantages: (1) the problems of section loss and entrapment of stain between section and slide during staining are eliminated, (2) relatively high staining temperature, akalinity, and alcohol concentration of the stain solvent (all of which promote loosening of pm-affixed sections from slides during staining) is allowed, and (3) staining is more even and selective. The procedure has been found to be reliable and fast enough to be of value in a significant variety of routine light microscope studies.     

Producing Frozen Sections of Calcified Bone

We describe a procedure for the rapid production and maintenance of fresh frozen bone biopsies which can be used for a variety of immunohistochemical techniques. Within 5 min of excision. tissue is placed in cold 5% polyvinyl alcohol, surrounded with 3% carboxymethylcel-lulose in a hand made aluminum foil embedding mold and frozen by immersion in an absolute ethanol/dry ice slurry at -70 C. The tissue block is attached to the specimen stub with cryocom-pound and installed in a -32 C cryostat whose tungsten carbide D profile knife is maintained at -70 C. Automatic controls are set at a slow cutting speed and the “sectioning window” is adjusted to fit the biopsy size. Knife angle, thickness gauge and antiroll bar are changed to produce a complete section. The block face is smoothly “papered” with a polyvinylpyrrolidone (PVP) impregnated Ross lens paper strip. A single section is cut and positioned on a sequentially numbered, acid cleaned, double dipped chrome-alum gelatin coated slide: adhesion is aided by “press-blotting” with bibulous paper. Sections are stored at -20 C or in a desiccator at room temperature. A brief fixation followed by removal of the water soluble PVP and lens paper generates fresh frozen bone sections suitable for further analysis.     

A method for the preparation of serial thick sections of plastic-embedded plant tissues.

A procedure is described in which thick sections (2-10 mu or more) of plastic-embedded plant tissues are mounted in serial order on slides for use in routine light microscopy. Sections are cut with a steel knife on a rotary microtome while the block and blade are bathed with 40% alcohol. The cut sections are placed, in order, in 50% alcohol in the small wells of modified plastic trays where they become flat, pliable and suitable for subsequent handling. Sections remain separate and in correct order in the trays while they are stained, washed, and prepared for final mounting on slides. Mounting involves a simple and rapid procedure of transferring the sections to a slide and heating first on a 70-75 C hot plate (to slowly evaporate the water around the section and to partially affix the section) and then on a C hot plate. This second heating ensures adhesion when xylene-base mounting media, which tend to loosen weakly adhered plastic from the slides, are used. The technique of staining the sections loose provides the following advantages: (1) the problems of section loss and entrapment of stain between section and slide during staining are eliminated, (2) relatively high staining temperature, alkalinity, and alcohol concentration of the stain solvent (all of which promote loosening of pre-affixed sections from slides during staining) is allowed, and (3) staining is more even and selective. The procedure has been found to be reliable and fast enough to be of value in a significant variety of routine light microscope studies.     

Cement Line Staining in Undecalcified Thin Sections of Cortical Bone

A technique for demonstrating cement lines in thin, undecalcified transverse sections of cortical bone has been developed. Cortical bone samples are processed and embedded undecalcified in methyl methacrylate plastic. After sectioning at 3-5 μm, cross-sections are transferred to a glass slide and flattened for 10 min. Sections of cortical bone are stained for 20 sec free-floating in a fresh solution of 1% toluidine blue dissolved in 0.1% formic acid. The section is dehydrated in t-butyl alcohol, cleared in xylene, and mounted with Eukitt's medium. Reversal lines appear as thin, scalloped, dark blue lines against a light blue matrix, whereas bone formation arrest lines are thicker with a smooth contour. With this technique cellular detail, osteoid differentiation, and fluorochrome labels are retained. Results demonstrate the applicability of a one-step staining method for cement lines which will facilitate the assessment of bone remodeling activity in thin sections of undecalcified cortical bone.     

Cement line staining in undecalcified thin sections of cortical bone.

A technique for demonstrating cement lines in thin, undecalcified transverse sections of cortical bone has been developed. Cortical bone samples are processed and embedded undecalcified in methyl methacrylate plastic. After sectioning at 3-5 microns, cross-sections are transferred to a glass slide and flattened for 10 min. Sections of cortical bone are stained for 20 sec free-floating in a fresh solution of 1% toluidine blue dissolved in 0.1% formic acid. The section is dehydrated in t-butyl alcohol, cleared in xylene, and mounted with Eukitt's medium. Reversal lines appear as thin, scalloped, dark blue lines against a light blue matrix, whereas bone formation arrest lines are thicker with a smooth contour. With this technique cellular detail, osteoid differentiation, and fluorochrome labels are retained. Results demonstrate the applicability of a one-step staining method for cement lines which will facilitate the assessment of bone remodeling activity in thin sections of undecalcified cortical bone.     

Cement line staining in undecalcified thin sections of cortical bone

A technique for demonstrating cement lines in thin, undecalcified, transverse sections of cortical bone has been developed. Cortical bone samples are processed and embedded undecalcified in methyl methacrylate plastic. After sectioning at 3-5 microns, cross-sections are transferred to a glass slide and flattened for 10 min. Sections of cortical bone are stained for 20 sec free-floating in a fresh solution of 1% toluidine blue dissolved in 0.1% formic acid. The section is dehydrated in t-butyl alcohol, cleared in xylene, and mounted with Eukitt's medium. Reversal lines appear as thin, scalloped, dark blue lines against a light blue matrix, whereas bone formation arrest lines are thicker with a smooth contour. With this technique cellular detail, osteoid differentiation, and fluorochrome labels are retained. Results demonstrate the applicability of a one-step staining method for cement lines which will facilitate the assessment of bone remodeling activity in thin sections of undecalcified cortical bone.     

A Basic Fuchsin and Alkalinized Methylene Blue Rapid Stain for Epoxyembedded Tissue

Sections 1 μ thick of epoxy-embedded, OsO₄-fixed tissues were stained with 4% aqueous basic fuchsin at 70 C for 1 min, rinsed well and destained, also at 70 C, for 1 min. A 2% aqueous methylene blue solution, alkalinized to pH 12.5 by mixing 1 N NaOH with the dye on the slide in the proportion of about 2:1, was then allowed to act for 2 min at 23-27 C. The stain was rinsed off the slide, and the preparation air dried before applying a mounting medium and cover glass. The mounting medium consisted of immersion oil sealed with epoxy household cement. Stains had not faded after 1 yr. The method is simple, rapid (total time 4-5 min), and provides sharp contrast between cellular and connective tissue components.     

A novel tissue array technique for high-throughput tissue microarray analysis — microarray groups

Tissue microarrays are ordered arrays of hundreds to thousands of tissue cores in a single paraffin block. We invented a novel method to make a high-throughput microarray group. Conventional smaller tissue microarrays were made first and then sectioned. Separate paraffin films were arrayed orderly onto a regular-sized glass slide to form a larger microarray group. Sections were not floated in a water bath but, rather, were cut singly using conventional microtome, arrayed orderly onto the glass slide with forceps instead of using a tape-based tissue transfer system, and then unfolded with warm water (46° C) using a micropipette. This not only lowers the difficulty in sectioning but the overall tissue disks can be included in the same section. A microarray group of 2,534 small disks (theoretically, 2,560 disks can be made; 26 fell off during the procedure), the most up to now, was successfully made and may be used in immunohistochemistry, mRNA in situ hybridization, and flourescent in situ hybridization.     

Fink-Heimer Silver Impregnation of Degenerating Axons and Terminals in Mounted Cryostat Sections of Fresh and Fixed Brains

The silver impregnation method of Fink and Heimer (Brain Res., 4: 369-74, 1967) has been used on cryostat sections of both fresh frozen and formalin-fixed brain tissue mounted on slide. The fixed brains were soaked in 25% sucrose for 2-3 days before freezing. The slides used for mounting were dipped in a 0.5% aqueous gelatin solution containing 50 mg of chrome-alum per 100 ml, drained and allowed to dry in a vertical position. Sections of fresh tissue were fixed for 16 hr in a 10% formalin solution buffered with phosphates to pH 7.0. Staining was carried out according to the Fink-Heimer procedure II and gave results comparable to those obtained on unmounted frozen sections of formalin-fixed material     

An Acetic Acid Dissociation, Air-Drying Technique for Insect Chromosomes, With Aceto-Lactic Orcein Staining

The method differs from mammalian techniques for somatic chromosomes in that it uses very small amounts of material. Drosophila melanogaster and an ant, Dorymyrmex sp., are used as examples. Pretreatment with 0.05% Colcemid in insect Ringer solution is applied to mature Drosophila larvae for 5 hr, by feeding, but Dorymyrmex prepupae require puncture and a 15 hr exposure of the puncture to the solution. Organs are removed under 1% sodium citrate, tansferred to fresh citrate for 10-20 min, than fixed in acetic-methanol, 1:3, for 30 min. Transfer to a drop of 60% acetic acid on a clean warmed slide dissociates the cells, which are spread by adding a small drop of fixative and tilting the slide in all directions. After immersion in acetic ethanol, 1:3, for 4 hr, rinsing in the stain solvent and draining the slides then have 2-3 drops of aceto-lactic orcein placed on each, coverslips added, and warmed (at about 50 C) for about 12 hr or until staining is sufficient. They can then either be treated as semipermanent or made permanent by allowing the coverslips to slide off in acetic-ethanol, dehydrating, and mounting in Euparal, or a synthetic resin.     

Fink-Heimer Silver Impregnation of Degenerating Axons and Terminals in Mounted Cryostat Sections of Fresh and Fixed Brains

The silver impregnation method of Fink and Heimer (Brain Res., 4: 369-74, 1967) has been used on cryostat sections of both fresh frozen and formalin-fixed brain tissue mounted on slide. The fixed brains were soaked in 25% sucrose for 2-3 days before freezing. The slides used for mounting were dipped in a 0.5% aqueous gelatin solution containing 50 mg of chrome-alum per 100 ml, drained and allowed to dry in a vertical position. Sections of fresh tissue were fixed for 16 hr in a 10% formalin solution buffered with phosphates to pH 7.0. Staining was carried out according to the Fink-Heimer procedure II and gave results comparable to those obtained on unmounted frozen sections of formalin-fixed material     

Sticky Wax Infiltration in the Preparation of Sawed Undecalcified Bone Sections

The undecalcified bone specimen was surfaced by an ordinary motor-driven circular saw. After thorough drying in air, the specimen was infiltrated with melted Caulk sticky wax (L. D. Caulk Co., Milford, Del., 19963) without casting in a block. The specimen was affixed to the Gillings-Hamco thin-sectioning machine with cut surface parallel to the circular diamond blade. Prior to sawing each section, the specimen surface was blown dry and coated with a thin supporting layer of stick wax. The section was then attached to an albumen-coated glass slide with the newly cut surface facing the slide. After drying in room temperature, the slide was soaked in xylene to partially dissolve the sticky wax, and the loosened residue was removed subsequently by gentle brushing. The section was mounted and covered with a coverglass. Sections 50-100 μ thick were prepared satisfactorily by this method. The advantages of using sticky wax as an infiltration medium depend on its physical properties: it is gluey when melted, and holds the bony trabeculae together; it becomes hard and nonsticky at room tempperature, and can be sawed together with bone tissue. Since a new layer of wax blends readily with the old wax surface, it allows the important supportive coating of wax to be added to the sawing surface for each section cutting     

Preparation of Leaf Epidermis for Topographic Study

Epidermal strips of leaves of the Gramineae can be prepared using the following technique: The mature leaf is dipped in boiling water to kill the cells, and decolorized in boiling 70% alcohol. It is cleared and softened in 88% lactic acid. Epidermal, mesophyll and vascular tissue is removed from a selected constant area of the leaf leaving an epidermal strip 1-3 cm in length. This is inverted on a slide, stained in lactopheno-cotton blue, and destained in 88% lactic acid. Transverse and longitudinal sections of the strip are obtained at this stage. The epidermal strip is finally mounted on a slide in 88% lactic acid. The preparation is photographed with a 35 mm camera using transmitted light, and a yellow filter in the microscope lamp. Photomicrographs of known enlargement are then prepared from which accurate measurements can be recorded. The technique is applicable to both fresh and herbarium material.     

A new method for preparation of undecalcified bone sections

A new method for preparation of sections of undecalcified bone is described. Samples of ovine bone were embedded in methylmethacrylate and thick-sectioned with a cutoff machine or commercial band saw. Composite slides were prepared by gluing white acrylic to glass using cyanoacrylate glue. Bone sections were glued to the composite slide and then surface polished by grinding or ultramilling. The polished surface of the section was then etched and stained. The techniques described in this paper reduce the time spent grinding or milling sections and improve resolution of surface-stained features of undecalcified bone sections.     

A New Method for Preparation of Undecalcified Bone Sections

A new method for preparation of sections of undecalcified bone is described. Samples of ovine bone were embedded in methylmethacrylate and thick-sectioned with a cutoff machine or commercial band saw. Composite slides were prepared by gluing white acrylic to glass using cyanoacrylate glue. Bone sections were glued to the composite slide and then surface polished by grinding or ultramilling. The polished surface of the section was then etched and stained. The techniques described in this paper reduce the time spent grinding or milling sections and improve resolution of surface-stained features of undecalcified bone sections.     

Autoradiography of Water-Diffusible Substances in Sections of Whole Baby Rats

Rats, 7 days postnatal which had been injected with a radioactive nuclide, were quick frozen and sectioned in the frozen state. An adhesive cellulose tape (Sellotape) was used to support the section during cutting, through freeze-drying, and attaching to slides. Dehydration of the frozen sections consisted of 1 hr in a chilled desiccator containing silica gel, then at reduced pressure of 2-3 mm Hg until quite dry. The exposed side of the section was sprayed with celloidin dissolved in amyl acetate and allowed to dry. This side of the section was attached to a slide, previously coated with 1% gelatin containing 0.1% chrome alum, by means of an adhesive consisting of 4% gelatin and 5% formalin in 60% glycerol. In applying this adhesive it is mandatory that a border of about 3 mm of bare glass be left outside the adhesive, to allow intimate contact between the sticky side of the tape and the glass. The adhesive was allowed to set for 20 min, the slide immersed in water lor 50 sec, and the cellulose layer of the tape peeled off. The rubber base from the tape was removed with chloroform, the slide dried, and the exposed surface of the section coated with celloidin in amyl acetate, by dipping. After this treatment, the slides could be coated by dipping in autoradiographic emulsion without affecting water-soluble radioactive substances in the tissue.     

An Abrasive Paper Technique for Preparing Sections of Wood for Microscopic Study

Dry wood specimens are sawed to 2mm thickness and impregnated with resin such as Lewisol 28 (Hercules Powder Co.). One side of the specimen is ground by hand on abrasive papers of grades #100, #180, #240, and #320. This side is then cemented to a petrographic glass slide with stick shellac and the other side similarly ground. Scratches can be eliminated by scraping the ground surface with the sharp edge of an ordinary glass microscopic slide. The section is removed by heating the slide, dissolving the shellac with alcohol and the resin of the embedding matrix with xylene. The sections can be stained in a hot saturated alcoholic solution of safranin O, counterstained with 0.01% fast green in an equal parts mixture of clove oil, methyl cellosolve, and absolute alcohol, and mounted in balsam.