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.     

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.     

Polyester Wadding for Specimen Orientation During Embedding in Methacrylates

Polyester fibers are not dissolved by either glycol methacrylate or methyl methacrylate. Commercial polyester wadding is consequently an advantageous material to use in getting precise orientation of tissue specimens during embedding in methacrylate.     

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.     

Alkaline and Acid Phosphatase Demonstration in Human Bone and Cartilage: Effects of Fixation Interval and Methacrylate Embedments

Human bone and cartilage specimens were evaluated for acid and alkaline phosphatase localization following varying fixation periods for fresh or frozen tissue. Formalin fixations of up to 183 hr were followed by embedment in methyl methacrylate; frozen tissue was examined either without fixation or following fixation for up to 1 hr and subsequent glycol or methyl methacrylate embedding. The humeral epiphysis of a young patient with osteogenic sarcoma showed optimum acid and alkaline phosphatase localization following fixation for periods up to 15 hr and embedding in methyl methacrylate. Frozen costochondral junction from a newborn with osteogenesis imperfecta type II showed optimum acid and alkaline phosphatase localization following 30 min fixation in formalin and embedding in methyl methacrylate or after 5 min fixation and embedding in glycol methacrylate.     

Alkaline and acid phosphatase demonstration in human bone and cartilage: effects of fixation interval and methacrylate embedments

Human bone and cartilage specimens were evaluated for acid and alkaline phosphatase localization following varying fixation periods for fresh or frozen tissue. Formalin fixations of up to 183 hr were followed by embedment in methyl methacrylate; frozen tissue was examined either without fixation or following fixation for up to 1 hr and subsequent glycol or methyl methacrylate embedding. The humeral epiphysis of a young patient with osteogenic sarcoma showed optimum acid and alkaline phosphatase localization following fixation for periods up to 15 hr and embedding in methyl methacrylate. Frozen costochondral junction from a newborn with osteogenesis imperfecta type II showed optimum acid and alkaline phosphatase localization following 30 min fixation in formalin and embedding in methyl methacrylate or after 5 min fixation and embedding in glycol methacrylate.     

Polyester wadding for specimen orientation during embedding in methacrylates

Polyester fibers are not dissolved by either glycol methacrylate or methyl methacrylate. Commercial polyester wadding is consequently an advantageous material to use in getting precise orientation of tissue specimens during embedding in methacrylates.     

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.     

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.     

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.     

Lymphocyte membrane antigens in glycol methacrylate embedded tissue

The use of formalin or Michel's solution either alone or in combination with acetone, and acetone, methanol or ethanol alone as fixatives, and glycol methacrylate as embedding medium were evaluated for their suitability in procedures to detect lymphocyte membrane antigens by OKT and Leu monoclonal antibodies in human tonsils. No staining was detected in sections fixed in 70% or absolute ethanol and embedded in glycol methacrylate with either the direct immunofluorescence or avidin-biotin methods. Fixation in Michel's solutions plus acetone at room temperature revealed staining by both. Neither method resulted in staining after fixation in Michel's solution plus acetone at 4 C presumably due to the slow action of the fixative. Staining was enhanced using a combination of primary and secondary biotinylated antibodies. Dual staining allowed concurrent detection of two antigens in the same section. Glycol methacrylate embedding is a possible replacement for ultracold storage in the preservation of tissue for immunofluorescent staining.     

Lymphocyte Membrane Antigens in Glycol Methacrylate Embedded Tissue

The use of formalin or Michel's solution either alone or in combination with acetone, and acetone, methanol or ethanol alone as fixatives, and glycol methacrylate as embedding medium were evaluated for their suitability in procedures to detect lymphocyte membrane antigens by OKT and Leu monoclonal antibodies in human tonsils. No staining was detected in sections fixed in 70% or absolute ethanol and embedded in glycol methacrylate with either the direct immunofluorescence or avidin-biotin methods. Fixation in Michel's solutions plus acetone at room temperature revealed staining by both. Neither method resulted in staining after fixation in Michel's solution plus acetone at 4 C presumably due to the slow action of the fixative. Staining was enhanced using a combination of primary and secondary biotinylated antibodies. Dual staining allowed concurrent detection of two antigens in the same section. Glycol methacrylate embedding is a possible replacement for ultracold storage in the preservation of tissue for immunofluorescent staining.     

Glycol Methacrylate Embedding of Algmate-Polylysine Microencapsulated Pancreatic Islets

A method for processing and embedding alginate-polylysine microencapsulated pancreatic tissue in glycol methacrylate resin (GMA) is described. Fixation in 4% phosphate buffered formaldehyde, processing in ascending concentrations of glycol methacrylate monomer and embedding in Technovit 7100 results in well preserved morphological details of hydrogels, hydrogel-cell interfaces, and encapsulated pancreatic tissue. Routine staining with Loeffler's methylene blue, hematoxylin and eosin, and Romanovsky-Giemsa gave excellent images of the GMA embedded alginate polylysine membrane and tissues allowing cells on the outside of the capsule to be analyzed effectively as part of the foreign body reaction against the capsule membrane.     

Glycol Methacrylate Embedding of Algmate-Polylysine Microencapsulated Pancreatic Islets

A method for processing and embedding alginate-polylysine microencapsulated pancreatic tissue in glycol methacrylate resin (GMA) is described. Fixation in 4% phosphate buffered formaldehyde, processing in ascending concentrations of glycol methacrylate monomer and embedding in Technovit 7100 results in well preserved morphological details of hydrogels, hydrogel-cell interfaces, and encapsulated pancreatic tissue. Routine staining with Loeffler's methylene blue, hematoxylin and eosin, and Romanovsky-Giemsa gave excellent images of the GMA embedded alginate polylysine membrane and tissues allowing cells on the outside of the capsule to be analyzed effectively as part of the foreign body reaction against the capsule membrane.     

Enzyme histochemistry on freeze-substituted glycol methacrylate-embedded tissue

We developed a method for histochemical demonstration of a wide range of enzymes in freeze-substituted glycol methacrylate-embedded tissue. Tissue specimens were freeze-substituted in acetone and then embedded at low temperature in glycol methacrylate resin. All enzymes studied (oxidoreductases, hydrolases) were readily demonstrated. The enzymes displayed high activity and were accurately localized without diffusion when tissue sections were incubated in aqueous media, addition of colloid stabilizers to the incubating media not being required. Freeze-substitution combined with low-temperature glycol methacrylate embedding permits the demonstration of a wide range of enzymes with accurate enzyme localization, maintenance of enzyme activity, and excellent tissue morphology.     

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.     

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 tisssues. 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.     

Spatially well-defined binary brushes of poly(ethylene glycol)s for micropatterning of active proteins on anti-fouling surfaces

We report a novel method for micropatterning of active proteins on anti-fouling surfaces via spatially well-defined and dense binary poly(ethylene glycol)s (PEGs) brushes with controllable protein-docking sites. Binary brushes of poly(poly(ethylene glycol) methacrylate-co-poly(ethylene glycol)methyl ether methacrylate), or P(PEGMA-co-PEGMEMA), and poly(poly(ethylene glycol)methyl ether methacrylate), or P(PEGMEMA), were prepared via consecutive surface-initiated atom transfer radical polymerizations (SI-ATRPs) from a resist-micropatterned Si(100) wafer surface. The terminal hydroxyl groups on the side chains of PEGMA units in the P(PEGMA-co-PEGMEMA) microdomains were activated directly by 1,1'-carbonyldiimidazole (CDI) for the covalent coupling of human immunoglobulin (IgG) (as a model active protein). The resulting IgG-coupled PEG microdomains interact only and specifically with target anti-IgG, while the other PEG microregions effectively prevent specific and non-specific protein fouling. When extended to other active biomolecules, microarrays for specific and non-specific analyte interactions with a high signal-to-noise ratio could be readily tailored.