An Embedding Method for Histochemical Studies of Undecalcified Skeletal Growth Plate

We have used glycol methacrylate to study undecalcified skeletal growth plate and subchondral bone. Minor modifications of the original technique including dehydration in glycol methacrylate vacuum infiltration and polymerization in the cold make it quite suitable for embedding of such tissues. Moreover, specimens can be processed quickly and the morphologic and biochemical integrity of the tissue retained so that histochemical procedures can be readily applied. Collagen, glycosaminoglycan, glycogen, lipid, calcium and the activity of alkaline and acid phosphatase were localized. This technique appears to be very useful for studying skeletal tissues.     

Ultramicrotome Chucks for Flat Castings

Paraformaldehyde-induced fluorescence in frozen-dried tissues survives embedding in glycol methacrylate. After freeze-drying and treatment with paraformaldehyde vapor, tissues to be examined by this technique are immersed in glycol methacrylate and placed in a dessicator which is then evacuated. They are usually left overnight in the dark; next day, the polymerizer is added and the tissues are again left overnight in the dark in the evacuated dessicator; for smaller blocks or certain tissues, these times can be shortened. The blocks are cut on a JB-4 microtome. Sections of 1-10μ can be made readily with a dry glass knife according to standard procedures.     

Glycol Methacrylate Sections of Frozen-Dried Tissues for Histofluorescence

Paraformaldehyde-induced fluorescence in frozen-dried tissues survives embedding in glycol methacrylate. After freeze-drying and treatment with paraformaldehyde vapor, tissues to be examined by this technique are immersed in glycol methacrylate and placed in a dessicator which is then evacuated. They are usually left overnight in the dark; next day, the polymerizer is added and the tissues are again left overnight in the dark in the evacuated dessicator; for smaller blocks or certain tissues, these times can be shortened. The blocks are cut on a JB-4 microtome. Sections of 1-10μ can be made readily with a dry glass knife according to standard procedures.     

Initiating Polymerization of Glycol Methacrylate with Cyclic Diketo Carbon Acids

Several pharmacologically active cyclic diketone carbon acids, including phenylbutazone and 2-phenyl-1,3-indandione, catalyze the polymerization of glycol methacrylate momomers. GMA-cyclic diketone carbon acid monomer mixtures incorporating imidazole polymerize smoothly without obvious exothermicity at temperatures ranging from ambient to -5 C without the use of ultraviolet light. The only equipment required for this embedding technique is a refrigerator with a freezing compartment which can maintain temperatures of -15 C. A recipe consisting of 5 ml glycol methacrylate (2-hydroxyethyl methacrylate), 0.8 ml 1-pentanol, 16 mg imidazole, and 30 mg monophenylbutazone is recommended for general use. The use of dicyclopentyl methacrylate-glycol methacrylate comonomer mixtures incorporating cyclic ketone catalysts is advocated, as blends of these monomers have low basophilia, and tissues embedded in these matrices stain sharply and brilliantly. It is hypothesized that the driving force for the cyclic ketone-mediated polymerization of glycol methacrylate under basic conditions is furnished by the lysis of cyclic ketone carbon acid peroxides.     

Initiating polymerization of glycol methacrylate with cyclic diketo carbon acids

Several pharmacologically active cyclic diketone carbon acids, including phenylbutazone and 2-phenyl-1,3-indandione, catalyze the polymerization of glycol methacrylate monomers. GMA-cyclic diketone carbon acid monomer mixtures incorporating imidazole polymerize smoothly without obvious exothermicity at temperatures ranging from ambient to -5 C without the use of ultraviolet light. The only equipment required for this embedding technique is a refrigerator with a freezing compartment which can maintain temperatures of -15 C. A recipe consisting of 5 ml glycol methacrylate (2-hydroxyethyl methacrylate), 0.8 ml 1-pentanol, 16 mg imidazole, and 30 mg monophenylbutazone is recommended for general use. The use of dicyclopentyl methacrylate-glycol methacrylate comonomer mixtures incorporating cyclic ketone catalysts is advocated, as blends of these monomers have low basophilia, and tissues embedded in these matrices stain sharply and brilliantly. It is hypothesized that the driving force for the cyclic ketone-mediated polymerization of glycol methacrylate under basic conditions is furnished by the lysis of cyclic ketone carbon acid peroxides.     

Neutralization of Acid in Glycol Methacrylate and the Use of Cyclohexanol as a Plasticizer

A method for neutralizing acidic compounds present in commercial samples of glycol methacrylate is described. Dimethylaminoethylmethacrylate is added to the glycol methacrylate, the amount required being determined by titration. Further improvement in staining of tissues embedded in the neutralized methacrylate can be brought about by the use of cyclohexanol as a plasticizer.     

The Staining Of Acid Fast Bacilli In Sections Of Glycol Methacrylate Embedded Tissues

Acid-fast bacilli can be stained in tissue embedded in glycol methacrylate. Modification of the Ziehl-Neelsen technique, along with changes in the formula of the plastic embedding medium, allow production of 1 to 2 micron sections which retain their integrity throughout the procedure, and within which the bacilli are clearly visible.     

The staining of acid fast bacilli in sections of glycol methacrylate embedded tissues.

Acid-fast bacilli can be stained in tissue embedded in glycol methacrylate. Modification of the Ziehl-Neelsen technique, along with changes in the formula of the plastic embedding medium, allow production of 1 to 2 micron sections which retain their integrity throughout the procedure, and within which the bacilli are clearly visible.     

Combined Lectin Binding and PAS/Alcian Blue Staining in Glycol Methacrylate Sections

Evaluation of cryofixation and paraffin and glycol methacrylate embedding showed that lectin binding was essentially independent of the embedding medium. Fluorescence intensity increased in the following order: glycol methacrylate, paraffin and cryostat sections, The optical resolution increased in the reverse order. Semi-thin glycol methacrylate sections provided satisfactory fluorescence intensities and the best resolution of all embedding techniques applied. Furthermore the lectin treated sections can be stained further using routine histological or specific histochemical methods. The potassium hy-droxide/alcian blue/periodic acid-phenylhydra-zine-Schiff method was used successfully to demonstrate sulfated and nonsulfated sialomucins. Lectins combined with mucin histochemistry allowed visualization of specific sugar residues in the same glycol methacrylate plastic section.     

Embedding Thin Plant Specimens for Oriented Sectioning

Small plant structures such as small primary roots, filamentous mosses and algae are difficult to orient for sectioning since they become wavy and curl during embedding. A method is described for embedding and orienting tiny plant specimens in a glycol methacrylate resin using self-constructed flat molds. Prior to sectioning, small samples can be oriented in both the longitudinal and the transverse plane. As several samples can be sectioned simultaneously, time-consuming trimming of the blocks is reduced substantially. The efficiency of this technique has been demonstrated using the tiny roots of the model plant Arabidopsis thaliana (L.) Heynh.     

The use of ultrasonication to remove non-specific precipitate

Summary The non-specific artifactual precipitate found in glycol methacrylate (GMA) embedded tissues, following acid phosphatase histochemical incubation, can now be eliminated with brief ultrasonication. A treatment of 1 to 2 min totally eliminated the artifactual residue, leaving the true histochemical reaction product and the physical integrity of the tissues intact. With this technique, most loosely bound precipitates, which clutter and mask GMA or conventionally embedded tissues, may now be treated and eliminated.     

The Application of Miller's Elastic Stain to Glycol Methacrylate Tissue Sections

The application of Miller's dilute elastic stain followed sequentially by Gill's III hematoxylin and a fast green counterstain produced a reliable and consistent method for differentially staining elastic fibers, nuclei, muscle and collagen in glycol methacrylate tissue sections. Evaluation of different methods of fixation and conditions of staining on animal tissue sections showed that elastic fibers in both perfusion and immersion fixed tissues can be intensely stained. The stability of Miller's elastic stain offers the potential of a commercially available histological stain reagent for coarse and fine elastic fibers in glycol methacrylate tissue sections.     

The application of Miller's elastic stain to glycol methacrylate tissue sections

The application of Miller's dilute elastic stain followed sequentially by Gill's III hematoxylin and a fast green counterstain produced a reliable and consistent method for differentially staining elastic fibers, nuclei, muscle and collagen in glycol methacrylate tissue sections. Evaluation of different methods of fixation and conditions of staining on animal tissue sections showed that elastic fibers in both perfusion and immersion fixed tissues can be intensely stained. The stability of Miller's elastic stain offers the potential of a commercially available histological stain reagent for coarse and fine elastic fibers in glycol methacrylate tissue sections.     

Assessment of Demyelination in Glycol Methacrylate Sections: A New Protocol for Cresyl Fast Violet Staining

A simple staining technique for nervous tissue is described. Tissue perfused with glutaraldehyde and formaldehyde and postfixed with osmium tetroxide is embedded in glycol methacrylate. One-micrometer sections are stained with 0.05% cresyl fast violet aqueous solution at 60 C for 5 min, washed with tap water and air dried. With this method the details of all nervous tissue elements are clearly demonstrated. The technique is particularly useful for assessing demyelination because the staining of axoplasm allows demyelinated axons to be well visualized.     

Assessment of demyelination in glycol methacrylate sections: a new protocol for cresyl fast violet staining

A simple staining technique for nervous tissue is described. Tissue perfused with glutaraldehyde and formaldehyde and postfixed with osmium tetroxide is embedded in glycol methacrylate. One-micrometer sections are stained with 0.05% cresyl fast violet aqueous solution at 60 C for 5 min, washed with tap water and air dried. With this method the details of all nervous tissue elements are clearly demonstrated. The technique is particularly useful for assessing demyelination because the staining of axoplasm allows demyelinated axons to be well visualized.     

Feminization of the forehead: contour changing to improve female aesthetics

Anthropologists have identified those characteristics which enable them to differentiate the male from the female skull. Some women have masculine skeletal characteristics which, if changed, would improve their facial appearance. Changing the skeletal configuration through bony contouring of the craniofacial skeleton for aesthetic purposes is a natural spinoff of craniomaxillofacial surgery. Techniques for sculpturing the masculine characteristics present in the foreheads of some females are discussed. The deformity has been divided into three subdivisions. Group 1 patients can be treated through bony contouring alone; group 2 patients require bony contouring in conjunction with a methyl methacrylate cranioplasty; and group 3 are those patients with a more severe deformity requiring osteotomies. The technique, results, complications, and patient acceptance are discussed.     

Hematoxylin Toluidine Blue-Phloxinate Staining of Glycol Methacrylate Sections of Retina and Other Tissues

For a detailed study of the developing chick retina a technique has been developed using glycol methacrylate embedding and a hematoxylin toluidine blue-phloxinate stain. After removal of the vitreous body, one half-segment of the eye is dehydrated through graded ethyl alcohols to 95%, infiltrated and embedded in glycol methacrylate, and sectioned at 2 μm. The sections are stained in alum hematoxylin and then in a mixture containing toluidine blue-phloxinate from a stock solution of the dye that has matured for 2-3 weeks. Differentiation is not required and there is only slight staining of the plastic matrix. The quality and clarity of the sections contrasts markedly with that of similarly stained 5 μm paraffin wax sections of the retina. This technique has also been applied to skin, spinal cord, dorsal root ganglia, pancreas and small intestine. The stained sections from these tissues have proved very useful in revealing structural components.     

Glycol methacrylate as an embedding medium for bone

A simple and reliable procedure for embedding undecalcified trabecular bone tissue in noncommercial glycol methacrylate (GMA) has been developed. The embedding mixture includes a monomer, methacrylic acid hydroxyethyl ester; a copolymer, methacrylic acid butyl ester; a cross-linker, ethylene glycol dimethacrylate; a catalyst, Luperco; a chemical initiator (N,N-dimethylaniline) and, to avoid excessive elevation of temperature during polymerization, a heat moderator, alpha-terpinene. The appropriate proportions of these components have been selected to give specimens which can be easily sectioned with classical microtomes and which do not swell but spread evenly on a water surface. Since polymerization occurs at -4 C, the method allows demonstration of such enzymatic activities as acid and alkaline phosphatase and carbonic anhydrase. It provides excellent preservation of bone tissue and in studies of bone metabolism allows histomorphometry as well as visualization of fluorescent labeling and radioactive markers. The cost is significantly less than available commercial kits. In our hands glycol methacrylate is at present more useful than methyl methacrylate and is used in our laboratory for routine embedding of bone tissue.     

Adaptations of Goldner's Masson Trichrome Stain for the Study of Undecalcified Plastic Embedded Bone

Specialized adaptations for application of Goldner's Masson trichrome stain to plastic embedded undecalcified bone specimens are presented. This stain can be used successfully on methyl-glycol methacrylate, glycol methacrylate and Spurr embedded bones. The stain affords the advantage of good cellular staining due to the hematoxylin component with concomitant sharp discrimination of mature bone matrix which stains green, immature new bone matrix which stains red, and calcified cartilage which stains very pale green. Use of red filters during photomicrography aids in bone-osteoid discrimination in black and white photographs.     

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