Method for histological preparation of bone sections containing titanium implants

A thin sectioning technique involving hand grinding has been developed to produce 20-40-microns-thick sections of bone-titanium implant sites. Components include: 1) surface staining of sections prior to mounting on slides so bone labels (oxytetracycline-HCl and 2,4-bis(N,N-dicarbomethyl)aminomethylfluorescein (DCAF] can be seen in sections viewed with transmitted light, 2) a pneumatic sample press for bonding sections to slides with a thin, uniform glue line and without trapped air bubbles, and 3) bonding methyl methacrylate embedded sections to clear acrylic slides with methyl methacrylate monomer to provide enhanced bond strength and grinding properties compared to those obtainable with glass slides. Sample cracking and distortion is minimized and the tissue-implant interface can be kept intact. The expense of start-up equipment for this technique is minimal.     

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 Method for the Preparation of Undecalcified Bone Sections for Light Microscopy and Microradiography

A method which gives good quality 1-2 μm thick sections of undecaldfied cancellous and thin cortical bones for light miuoscopy is described. Formalin fixed material is dehydrated in graded acetones and embedded in a modiEed formula of Spurr's low viscosity embedding medium. After a 16 hour polymerisation period at 60 C, sections are cut at 1-2 μm thickness on a Porter-Blum JB4A rotary microtome Using glass knives. Sections are attached to clean glass slides with heat, the resin degraded in bromine vapour and removed in acetone. This allows comparative ease of staining. The technique is rapid, does not interfere with tetracycline fluorescence and the same specimens can be used to prepare thick sections for microradiography.     

The Membrane Method of Electron Microscope Autoradiography

Thin sections of methacrylate and Araldite embedded tissues labelled with radioactive isotopes were transferred with a wire loop or brush from the knife edge onto thin formvar membranes which covered 7 mm holes in 76 × 25 × 1.5 mm or 76 × 38 × 1.5 mm plastic slides. To facilitate the mounting of sections, a platform supported the plastic slides close to the ultramicrotome knife. Photographic emulsion diluted 1:5 or 1:10 with water was applied with a pipette to the upper surface of each formvar membrane to cover the mounted sections. Excess emulsion was drained off and the remaining thin film was dried on a warm plate at 45 C to produce a uniform layer over the sections. After storing in the dark for several weeks, preparations were processed in photographic solutions and washed, and sometimes stained, before applying electron microscope grids to the underside of each formvar membrane. To detach each grid with its adherent formvar, section and emulsion, the membrane was pierced around the perimeter of the grid. Grain counts made over nuclei of cells labelled with tritiated thymidine indicate that emulsion is uniformly distributed over each section and that quantitative comparison is possible between labelled areas.     

Improvement of the Teflonized Slides Used in the Immunofluoresgent Antibody Technique

The indirect fluorescent antibody (IF) test is often performed on glass slides prepared with smears or tissue sections containing an antigen. It is convenient to have several separate and distinct areas of antigen on the same slide, e.g. to compare dilution steps, and to save time when producing the antigen slides. With this end in view a technique for preparing slides with wells in a thin film of teflon-like compound has been elaborated (Goldman 1968). This technique is useful when a great number of slides need to be prepared in advance, e.g. to test a substantial amount of material. It has, however, not been widely used im serology for Babesia, perhaps because any teflon remaining within the wells is stained with the fluorescent dye, thus creating a potentially disturbing background.     

Double Emulsion Autoradiography for Identifying Tritium-Labelled cells in sections

The sensitivity of tissue autoradiography can be doubled and the number of false negative cells nearly eliminated by interposing thin tissue sections between two layers of photographic emulsion. A mouse was given 50 μc of tritiated thymidine (SA 2,500 c/M) intraperitoneally and killed 1.5 hr later. A portion of the small bowel was removed, fixed and embedded in methacrylate in the usual way. Sections 2 μ thick were cut and allowed to flatten on water at 40° C. Some sections were used to make conventional single emulsion auto-radiographs and other sections were interposed between two layers of emulsion by first coating slides with NTB 3 emulsion, picking up the sections from a water bath at 18° C, drying, soaking 1 min in benzene, drying, and then dipping again in NTB 3 emulsion. They were exposed at 4° C in a low humidity, 100% CO₂ atmosphere for 10 days, developed and covered in the usual way. There was an average of 20.16 ± 1.4 grains per labelled cell in the double emulsion group compared with 10.6 ± 0.9 grains in the single emulsion group. In the double emulsion autoradiographs there were 55.1 ± 1.65 labelled cells per unit area as compared with 39.8 2 2.0 in the single emulsion group.     

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     

Microwave-aided technique to detect bromodeoxyuridine in S-phase cells using immunogold-silver staining and plastic-embedded sections

Summary A technique is described to detect bromodeoxyuridine (BrdU) incorporate by cells in S-phase, with a monoclonal antibody, using removable plastic embedding and immunogold-silver staining (IGSS). The incubation times were reduced and the immunological reactions enhanced by microwave irradiation.The embedding in methyl methacrylate enabled us to make thinner sections and it improved the quality of the preparations. The methyl methacrylate did not hinder the reaction of BrdU with the antibody because it could be removed prior to the IGSS procedure. The IGSS procedure appeared to be very sensitive, requiring lower concentrations of the antibodies than other methods. The use of microwave irradiation shortened the time needed to stain a section from 7 to less than 4 h. Furthermore, using microwave irradiation, the concentration of the antibodies needed could be reduced even further compared with the normal IGSS procedure.In sections of the mouse testis and small intestine only nuclei of cells known to be able to proliferate appeared BrdU positive. The non-specific background staining was found to be negligible. In testes of mice that received both³H-thymidine and BrdU more than 95% of the radioactively labelled cells also showed BrdU label and vice versa. This indicates that both methods are equally sensitive for detecting cells in S-phase.     

Some New Methods for Affixing Sections to Glass Slides. I. Aqueous Adhesives

As a new aqueous adhesive to affix sections to glass slides, hydrolyzed vinyl-triethoxysilane-either pure, in combination with polyvinyl alcohol or with specially prepared aqueous polyacrylate solutions-was applied. The silane proved to be very effective in enhancing bonding to the glass surface. As a general aqueous adhesive, a solution of 2% polyvinyl alcohol (m.w. 108,000; 99.7% hydrolyzed) with 0.2% hydrolyzed vinyltriethoxysilane is recommended. This stock solution is diluted 1:10 to 1:50 and used directly to float sections onto slides on a warming plate.     

Some new methods for affixing sections to glass slides. I. Aqueous adhesives

As a new aqueous adhesive to affix sections to glass slides, hydrolyzed vinyltriethoxysilane--either pure, in combination with polyvinyl alcohol or with specially prepared aqueous polyacrylate solutions--was applied. The silane proved to be very effective in enhancing bonding to the glass surface. As a general aqueous adhesive, a solution of 2% polyvinyl alcohol (m.w. 108,000; 99.7% hydrolyzed) with 0.2% hydrolyzed vinyltriethoxysilane is recommended. This stock solution is diluted 1:10 to 1:50 and used directly to float sections onto slides on a warming plate.     

A simplified technique for low temperature methyl methacrylate embedding

A simplified method for low temperature methyl methacrylate embedding with inhibited methyl methacrylate monomer is demonstrated using proper concentrations of benzoyl peroxide and N,N-dimethylaniline. The polymerized tissue blocks cut well and the tissue sections obtained show excellent acid phosphatase activity when demonstrated with the newly improved technique and Goldner's staining. Likewise, double tetracycline labels are well revealed by fluorescence microscopy.     

Embedding in Epoxy Resins for Ultrathin Sectioning in Electron Microscopy

Fixed tissue is dehydrated with tertiary butyl alcohol overnight. The following day it is cleared in toluene, infiltrated and embedded in Araldite resin-hardener-accelerator mixture without dibutyl phthalate, and polymerized at 60° C. More rapid than previous techniques, this method gives blocks which do not fracture unduly on trimming and provides sections of soft tissues at 1 μ for phase contrast microscopy, as well as ultrathin sections which cut as easily with glass knives as sections of methacrylate. Araldite manufactured in the U.S.A. and in England are different. Satisfactory proportions for the American are: hardener DDSA, 3.5 ml; casting resin 6005, 5.0 ml; accelerator B, 0.12 ml. For the British product, these are: hardener 964 B, 5.0 ml; casting resin M, 5.0 ml; accelerator 964 C, 0.25 ml. The use of 2% agar for orienting small specimens in Araldite is feasible. Mallory's borax-methylene blue has been applied to the staining of Araldite sections as thin as 0.5 μ mounted on glass slides.     

A Simplified Technique for Low Temperature Methyl Methacrylate Embedding

A simplified method for low temperature methyl methacrylate embedding with inhibited methyl methacrylate monomer is demonstrated using proper concentrations of benzoyl peroxide and N,N-dimethylaniline. The polymerized tissue blocks cut well and the tissue sections obtained show excellent acid phosphatase activity when demonstrated with the newly improved technique and Goldner's staining. Likewise, double tetracycline labels are well revealed by fluorescence microscopy.     

Histoautoradiographic Localization of Calcium in Oat Plant Tissues

The leaching of water-soluble and exchangeable calcium in histoautoradiog-raphy of oat tissue can be prevented by using acetone as the dehydration fluid (freeze substitution technique) and by keeping the tissue sections, while stretching on water, embedded in the methacrylate matrix. Ca⁴⁵ was either added to the mineral solution on which the oat plants were grown (75 μc), or applied on the leaf surface (8 μc). After freezing in melting isopentane, specimens of 1-2 mm dimensions are fixed for 24 hr in an acetone-OsO₄ (1%) solution at—80 C. Dehydration is obtained by transferring the material every day for 6 successive days to a fresh acetone solution at—80 C. The material is infiltrated by a three-time renewed monomer methacrylate mixture (methylmethacrylate I, butylmethacrylate 4) at—50 C. The specimens are embedded in the polymerizing methacrylate mixture at room temperature. Sections of 4-8 μ are easily cut with a rotating microtome. If the methacrylate is not removed from the sections, they can be stretched on water without leaching of calcium. The presence of methacrylate in no way hinders microscopic observation nor effective histoautoradiography.     

Methacrylate as an Embedding Medium for Woody Tissues

Methacrylate can be readily infiltrated into woody tissues. After infiltration, the tissue is transferred to a polymerizing mixture of 95:5 butyl methacrylate to methyl methacrylate by volume. For each 100 ml of polymerizing mixture, 2 grams of Luperco CDB catalyst are added. The hardness of the matrix may be increased by increasing the proportion of methyl methacrylate. Polymerization is accomplished by 2 days in a 50° C oven or 2 days in a small ultraviolet radiation chamber (42° C), the latter being the technique of choice. The blocks can be sectioned readily at 6 μ to more than 30 μ. Sectioning is facilitated by keeping the block wet with a 1:1 glycerol-alcohol solution. Best preparations are obtained when the matrix is removed after sectioning; however, staining in safranin O-fast green FCF may be-accomplished through the matrix. The technique is very rapid, convenient to use, and has produced excellent preparations from several species of woody plants.     

Some new methods for affixing sections to glass slides. II. Organic-solvent based adhesives

Adhesion of various organic-solvent based adhesives to glass slides could be greatly improved by first priming the slide with a copolymer of allyl methacrylate and methacryloxypropyltrimethoxysilane. The use of different solvents and types of adhesives with these slides is discussed. Cellulose nitrate in different esters of acetic acid proved to be an effective adhesive for varied sections at room temperature and in the cryostat. Carbowax sections as a special case preferably were affixed with polyisobutylene in petroleum ether. Most of the attachments formed resisted even boiling water.     

Some New Methods for Affixing Sections to Glass Slides. Ii. Organic-Solvent Based Adhesives

Adhesion of various organic-solvent based adhesives to glass slides could be greatly improved by first priming the slide with a copolymer of allyl methacrylate and methacryloxypropyltrimethoxysilane. The use of different solvents and types of adhesives with these slides is discussed. Cellulose nitrate in different esters of acetic acid proved to be an effective adhesive for varied sections at room temperature and in the cryostat. Carbowax sections as a special case preferably were affixed with polyisobutylene in petroleum ether. Most of the attachments formed resisted even boiling water.     

A rapid immunogold-silver staining for detection of bromodeoxyuridine in large numbers of plastic sections, using microwave irradiation

Summary A rapid and convenient method for the large scale, immunogold-silver staining (IGSS) of bromodeoxyuridine (BrdU) incorporated by S phase cells, by means of a monoclonal antibody (anti-BrdU) is described. Nineteen slides at a time can be incubated with the antibodies and the protein A-gold (PAG) in staining jars. The antibody and protein A-gold solutions could be used at least five times to incubate new batches of slides. The incubation times with these solutions were shortened by means of microwave irradiation. In this way 200 slides carrying at least 800 sections could be easily processed under the same conditions in one day, using 1.25ml neat antibody solutions of anti-BrdU and rabbit anti-mouse.For light microscopy bothpplastic embedding systems: methylmethacrylate (MMA) and glycolmethacrylate (GMA) can be stained with this technique. The MMA sections, of which the plastic has to be removed before the IGSS, has the advantage of a stronger labelling intensity. The GMA plastic, which contains a cross-linking, agent cannot be removed and consequently for GMA sections it is necessary to incubate the sections with a proteolytic enzyme (trypsin) before the IGSS, to reexpose the antigenic binding sides. However, the GMA sections can be allowed to air dry during the IGSS without negative effects on the morphology. This makes it possible to perform the antibody and the PAG-incubating steps on one day and to finish the IGSS the next day. In this way twice as many GMA slides can be incubated with the same antibody and PAG solutions than with MMA slides.In both plastic embedding systems the intensity of the BrdU labelling was found to be stronger in Carnoy's than in Bouin's fixed sections.