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.     

Simultaneous demonstration of bone alkaline and acid phosphatase activities in plastic-embedded sections and differential inhibition of the activities

Summary Bone alkaline (AlP) and acid phosphatase (AcP) activities were simultaneusly demonstrated in tissue sections obtained from mice, rats, and humans. The method involved tissue fixation in ethanol, embedding in glycol methacrylate (GMA), and demonstration of AlP and AcP activities employing a simultaneous coupling azo dye technique using substituted naphthol phosphate as a substrate. AlP activity was demonstrated first followed by AcP activity. Both enzyme activities were demonstrated in tissue sections from bones fixed and/or stored in acetone or 70% ethanol for up to 14 days or stored in GMA for 2 months. AlP activity in tissue sections from bones fixed in 10% formalin, 2% glutaraldehyde, or formal-calcium, however, was markedly inhibited after 3–7 days and was no longer detectable after 14 days of fixation. Moreover, AlP activity was diminished in tissue sections from bones fixed in 70% ethanol or 10% formalin and subsequently demineralized in 10% EDTA (pH7) for 2 days, and the activity was completely abolished in tissue sections from bones subsequently demineralized in 5% formic acid: 20% sodium citrate (1:1, pH 4.2) for 2 days. Methyl methacrylate (MMA) embedding at concentrations above 66% completely inhibited AlP activity. AcP activity, however, was only partially inhibited by formalin, glutaraldehyde, or formal-calcium after 7 or 14 days of fixation or by MMA embedding and was unaffected by the demineralizing agent formic acid-citrate for 2 days. While AcP activity was preserved in bones fixed in formalin and subsequently demineralized in EDTA, the activity was completely abolished when EDTA demineralization was carried out on bones previously fixed in 70% ethanol. These results indicate that bone AlP and AcP activities can be demonstrated simultaneously in the same section using a simple tissue preparation technique and that the activities are retained in tissues fixed and/or stored in acetone, 70% ethanol or GMA, but are differentially inactivated by other fixatives studied, and by EDTA, formic acid-citrate, and MMA embedding.Abbreviations AcP acid phosphatase - AlP alkaline phosphatase - GMA glycol methacrylate - MMA methyl methacrylate - EDTA ethylenediaminetetraacetic acid     

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     

Resin tissue microarrays: a universal format for immunohistochemistry.

Tissue microarray (TMA) technology allows the miniaturization and characterization of multiple tissue samples on a single slide and commonly uses formalin-fixed paraffin-embedded (FFPE) tissue or acetone-fixed frozen tissue. The former provides good morphology but can compromise antigenicity, whereas the latter provides compromised morphology with good antigenicity. Here, we report the development of TMAs in glycol methacrylate resin, which combine the advantages of both methods in one embedding format. Freshly collected tissue fixed in -20C acetone or 10% neutral buffered formaldehyde were cored and arrayed into an intermediary medium of 2% agarose before infiltration of the agarose array with glycol methacrylate resin. Acetone-fixed resin TMA demonstrated improved morphology over acetone-fixed frozen TMA, with no loss of antigenicity. Staining for extracellular, cell surface, and nuclear antigens could be realized with monoclonal and polyclonal antibodies as well as with monomeric single-chain Fv preparations. In addition, when compared with FFPE TMA, formalin-fixed tissue in a resin TMA gave enhanced morphology and subcellular detail. Therefore, resin provides a universal format for the construction of TMAs, providing improved tissue morphology while retaining antigenicity, allows thin-section preparation, and could be used to replace preparation of frozen and FFPE TMAs for freshly collected tissue.     

Immunofluorescent Labelling of K-Papovavirus Antigens in Glycol Methacrylate Embedded Material: A Method for Studying Infected Cell Populations by Fluorescence Microscopy and Histological Staining of Adjacent Sections

Trypsin and protease V (pronase) were studied for their ability to enhance immuno-fluorescent labelling of papovavirus antigens in glycol methacrylate embedded sections of organs infected with murine K-papovavirus. Treatment of Bouin's fixed sections with 0.4% trypsin for 30 minutes resulted in specific immunofluorescent staining equal to that seen in frozen sections and produced little if any loss of histological detail. Treatment with protease V resulted in less brilliant fluorescence and less satisfactory tissue preservation. Studies were then conducted to determine the fixative which would produce brightest specific fluorescent antibody staining of papovavirus-infected cells while providing clearest definition of intranuclear inclusions and best morphological detail in histologically stained adjacent sections. Brightest immunofluorescence staining was accomplished on material fixed in 96% ethanol/1% glacial acetic acid or Bouin's solution. These fixatives also gave clear definition of intranuclear inclusions with histological stains and provided excellent morphological detail. Phosphate buffered paraformaldehyde/picric acid and 3.7% formalin gave less satisfactory fluorescence and obscured intranuclear inclusions in histological preparations. Sections fixed in 4% paraformaldehyde, 4% paraformaldehyde/1% glutaraldehyde, and 0.5 M p-toluenesulfonic acid were negative for specific fluorescence. Glycol methacrylate, used with proper fixation and trypsin pretreatment of sections, provides a useful embedding medium for immunofluorescent identification of virus-infected cells, and the 1.0-2.0 μm sections routinely obtainable with GMA permit study of individual infected cells by fluorescent antibody and histological staining of adjacent sections.     

A Tribasic Stain for Thin Sections of Plastic-Embedded, OsO4-Fixed Tissues

Thin (0.5-1 μ) sections of plastic-embedded, OsO₄-fixed tissues were attached to glass slides by heating to 70 C for 1 min. A saturated solution combining toluidine blue and malachite green was prepared in ethanol (8% of each dye) or water (4% of each dye). Methacrylate or epoxy sections were stained in the ethanol solution for 2-5 min. The water solution was more effective for some epoxy sections (10-80 min). Epoxy sections could be mordanted by 2% KMnO₄, in acetone (1 min) before use of the aqueous dye, reducing staining time to 5-10 min and improving contrast. Aqueous basic fuchsin (4%) was used as the counter-stain in all cases; staining time varied from 1-30 min depending upon the embedding medium and desired effects, methacrylate sections requiring the least time. In the completed stain, nuclei were blue to violet; erythrocytes and mitochondria, green; collagen and elastic tissue, magenta; and much and cartilage, bright cherry red. Sections were coated with an acrylic resin spray and examined or photographed with an oil-immersion lens.     

Simultaneous demonstration of bone alkaline and acid phosphatase activities in plastic-embedded sections and differential inhibition of the activities

Bone alkaline (AlP) and acid phosphatase (AcP) activities were simultaneously demonstrated in tissue sections obtained from mice, rats, and humans. The method involved tissue fixation in ethanol, embedding in glycol methacrylate (GMA), and demonstration of AlP and AcP activities employing a simultaneous coupling azo dye technique using substituted naphthol phosphate as a substrate. AlP activity was demonstrated first followed by AcP activity. Both enzyme activities were demonstrated in tissue sections from bones fixed and/or stored in acetone or 70% ethanol for up to 14 days or stored in GMA for 2 months. AlP activity in tissue sections from bones fixed in 10% formalin, 2% glutaraldehyde, or formal-calcium, however, was markedly inhibited after 3-7 days and was no longer detectable after 14 days of fixation. Moreover, AlP activity was diminished in tissue sections from bones fixed in 70% ethanol or 10% formalin and subsequently demineralized in 10% EDTA (pH 7) for 2 days, and the activity was completely abolished in tissue sections from bones subsequently demineralized in 5% formic acid: 20% sodium citrate (1:1, pH 4.2) for 2 days. Methyl methacrylate (MMA) embedding at concentrations above 66% completely inhibited AlP activity. AcP activity, however, was only partially inhibited by formalin, glutaraldehyde, or formal-calcium after 7 or 14 days of fixation or by MMA embedding and was unaffected by the demineralizing agent formic acid-citrate for 2 days. While AcP activity was preserved in bones fixed in formalin and subsequently demineralized in EDTA, the activity was completely abolished when EDTA demineralization was carried out on bones previously fixed in 70% ethanol.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Embedding and Fixation Techniques for Immunohistochemical Staining with Anti-DNA Polymerase α and Ki-67 Monoclonal Antibodies to Analyze the Proliferative Potential of Tumors

Anti-DNA polymerase a and Ki-67 are monoclonal antibodies that recognize nuclear antigens expressed in proliferating cells. In this study, we evaluated various methods of embedding and fixing brain tumor specimens to optimize staining with these antibodies. In fresh frozen sections, postfixation with 4% paraformaldehyde, 100% methanol, 95% ethanol and 10% buffered formalin were tested; also tested were prefixation with 4% paraformaldehyde followed by freezing and fixation with 100% methanol, 95% ethanol, or 10% buffered formalin followed by embedding in paraffin. For both antibodies, postfixation of fresh frozen sections with 4% paraformaldehyde at 4 C gave the most intense staining and lowest background activity while preserving histological features. This technique can be used in routine clinical practice to predict the growth potential of tumors.     

Immunohistochemistry on Resin Sections: A Comparison of Resin Embedding Techniques for Small Mucosal Biopsies

We have modified resin embedding methods to provide optimal information from en-doscopic biopsies. Mucosal biopsies were fixed either in buffered formalin and processed for embedding in Araldite or in acetone containing protease inhibitors and embedded in glycol meth-acrylate (GMA). GMA embedding generated an im-munophenotypic profile similar to that obtained in frozen sections while yielding far superior morphology and greater numbers of sections from small biopsies. The phenotypic markers included those for T cells, macrophages, mast cells, eosin-ophils and neutrophils. We have also demonstrated collagens, cell adhesion molecules and integrin molecules. Sections of similar quality were obtained with Araldite but the repertoire of antibodies was restricted to those which can be applied to formalin fixed, paraffin embedded tissues. We suggest that for optimal results, small biopsies to be subjected to immunochemistry are fixed in acetone at -20 C with the inclusion of protease inhibitors and embedded in GUIA with careful temperature control.     

Freeze-drying of bone tissue: immunocytochemistry and enzyme histochemistry on paraffin embedded and low-temperature resin embedded specimens.

A simple protocol of tissue preparation was sought, which would enable marker enzymes of bone cells and extracellular matrix antigens to be localized in the same tissue section with high optical resolution. For this purpose, snap-frozen samples of rat fetal skeletal tissues were dried in a FDU 010 freeze-drying unit (Balzers) for 8-12 h at -50 to -40 degrees C and 0.02 bar. Freeze-dried tissues were either vacuum-infiltrated at 45 degrees C and embedded undemineralized in Paraplast, or vacuum-infiltrated overnight at 4 degrees C and embedded undemineralized in glycol methacrylate. These procedures enabled enzyme cytochemistry for alkaline phosphatase and tartrate-resistant acid phosphatase, and immunocytochemical staining for collagen types I, III, and laminin to be performed on the same sections. No pretreatment of the sections was necessary to reveal collagen antigenicity. This study reveals the possibility of complementing immunocytochemical studies of extracellular matrix with enzyme cytochemistry and, above all, with the excellent tissue preservation and high resolution afforded by plastic embedding.     

Immunohistochemical antigen demonstration in plastic-embedded lymphoid tissue

We describe a method for post-embedding immunohistochemical demonstration of a wide range of antigens in glycol methacrylate-embedded tissue. Rat spleen and thymus tissues were fixed by immersion in fixatives containing different concentrations of paraformaldehyde, washed in sucrose phosphate buffer, dehydrated in acetone, infiltrated in a glycol methacrylate mixture in which the commonly used softener 2-butoxyethanol was replaced by butaandiol monoacrylate, and embedded. Trypsin was used to re-expose the masked antigenicity. Excellent results were obtained with a panel of monoclonal antibodies (MoAbs) directed against T-cells, B-cells, Ia-positive cells, macrophages, follicular dendritic cells, and leucocyte common antigen-bearing cells. The method described combines exact localization of antigens with optimal tissue morphology.     

Immunoperoxidase detection of myeloid antigens in glycolmethacrylate-embedded human bone marrow

Different methods for fixation and exposure of antigenic determinants were tested for detection of a granulocytic differentiation antigen by the monoclonal antibody L12-2, using an indirect immunoperoxidase method on semi-thin sections of undecalcified, glycolmethacrylate-embedded human bone marrow biopsies. Fixation in Bouin's solution for 3 hr gave a more intense and more homogeneous immunological staining than fixation in absolute methanol, 4% formalin, B5, or Michel's medium, and the morphological detail was excellent. Digestion by pronase or trypsin was required. Coating the glass slides with Alcian blue prevented loss of sections from the slides during the staining procedure. Bouin fixation also made possible detection of two other differentiation antigens expressed in the granulocytic series, using the monoclonal antibodies 1G10 and R1B19. Furthermore, the same technique also permitted detection of factor VIII-RAg in the megakaryocytes, as well as recognition of cells of the erythroid series by use of polyclonal rabbit antisera.     

Enzyme histochemical demonstration of NADH dehydrogenase on resin-embedded tissue

We describe a method for enzyme histochemical demonstration of NADH dehydrogenase in cold (4 degrees C)-processed resin-embedded tissue. The effects on NADH dehydrogenase activity of processing tissue through a variety of dehydrating agents and embedding in three different acrylic resins were evaluated. The optimal procedure to maintain NADH dehydrogenase activity used a short (3-hr) fixation in 1% paraformaldehyde solution, followed by dehydration in acetone and embedding in glycol methacrylate resin. Embedding of tissue in resin combined preservation and accurate localization of NADH dehydrogenase activity with good tissue morphology. Blocks of the resin-embedded tissue could be stored at room temperature for at least 6 months without loss of NADH dehydrogenase activity.     

Embedding Iliac Bone Biopsies at Low Temperature using Glycol and Methyl Methacrylates

A mixture of pure and anhydrous glycol methacrylate and methyl methactylate is used as an embedding medium for iliac bone biopsies. Infiltration is carried out at -20 C with the embedding medium and a cold inactivated catalyst-initiator system. Raising the temperature to 4 C initiates polymerization and limits the peak temperature of polymerization to 25 C. In this way, such thermolabile enzymes as osteoclastic acid phosphatase are preserved. After staining, sections are dehydrated in polyethylene glycol 400 30% in 2-propanol. This gives flat sections and improves staining properties.     

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.     

Immunofluorescent labelling of K-papovavirus antigens in glycol methacrylate embedded material: a method for studying infected cell populations by fluorescence microscopy and histological staining of adjacent sections

Trypsin and protease V (pronase) were studied for their ability to enhance immunofluorescent labelling of papovavirus antigens in glycol methacrylate embedded sections of organs infected with murine K-papovavirus. Treatment of Bouin's fixed sections with 0.4% trypsin for 30 minutes resulted in specific immunofluorescent staining equal to that seen in frozen sections and produced little if any loss of histological detail. Treatment with protease V resulted in less brilliant fluorescence and less satisfactory tissue preservation. Studies were then conducted to determine the fluorescence and less satisfactory tissue preservation. Studies were then conducted to determine the fixative which would produce brightest specific fluorescent antibody staining of papovavirus-infected cells while providing clearest definition of intranuclear inclusions and best morphological detail in histologically stained adjacent sections. Brightest immunofluorescence staining was accomplished on material fixed in 96% ethanol/1% glacial acetic acid or Bouin's solution. These fixatives also gave clear definition of intranuclear inclusions with histological stains and provided excellent morphological detail. Phosphate buffered paraformaldehyde/picric acid and 3.7% formalin gave less satisfactory fluorescence and obscured intranuclear inclusions in histological preparations. Sections fixed in 4% paraformaldehyde, 4% paraformaldehyde/1% glutaraldehyde, and 0.5 M p-toluenesulfonic acid were negative for specific fluorescence. Glycol methacrylate, used with proper fixation and trypsin pretreatment of sections, provides a useful embedding medium for immunofluorescent identification of virus-infected cells, and the 1.0-2.0 micron sections routinely obtainable with GMA permit study of individual infected cells by fluorescent antibody and histological staining of adjacent sections.     

Science and Art in Preparing Tissues Embedded in Plastic for Light Microscopy, with Special Reference to Glycol Methacrylate, Glass Knives and Simple Stains

Plastic embedding preserves tissue structure much more faithfully than does paraffin. Acrylic polymerization is innocuous to dye-binding groups in sections. The water solubility of glycol methacrylate monomer and the hydrophilic properties of the polymer allow for convenience in dehydration and for versatility in staining sections. Five years of experience with glycol methacrylate (GMA) embedding for light microscopy is summarized. Methods for purifying GMA monomer are cited. Procedures for fixing, dehydrating, embedding, polymerizing, sectioning and staining, using GMA, are explained. A method is provided for making glass knives long enough to cut large blocks. Simple, reliable, quick staining methods are outlined. When compared with paraffin, GMA offers opportunities for simpler, quicker procedures and yields sections of superior quality, greater information content, and less distortion.     

Science and art in preparing tissues embedded in plastic for light microscopy, with special reference to glycol methacrylate, glass knives and simple stains.

Plastic embedding preserves tissue structure much more faithfully than does paraffin. Acrylic polymerization is innocuous to dye-binding groups in sections. The water solubility of glycol methacrylate monomer and the hydrophilic properties of the polymer allow for convenience in dehydration and for versatility in staining sections. Five years of experience with glycol methacrylate (GMA) embedding for light microscopy is summarized. Methods for purifying GMA monomer are cited. Procedures for fixing, dehydrating, embedding, polymerizing, sectioning and staining, using GMA, are explained. A method is provided for making glass knives long enough to cut large blocks. Simple, reliable, quick staining methods are outlined. When compared with paraffin, GMA offers opportunities for simpler, quicker procedures and yields sections of superior quality, greater information content, and less distortion.