Standardization of various applications of methacrylate embedding and silver methenamine for light and electron microscopy immunocytochemistry

Summary The use of butyl-methyl-methacrylate embedding and the application of the silver methenamine (SM) method as a poststaining of the immunoperoxidase-DAB (IP) procedure led to the standardization of several useful methods for the visualization of tissue antigents at the light and electron microscope level. These procedures included: 1) Standardization of the actual methacrylate embedding; 2) The IP-SM method with an without periodic acid oxidation, which provided 100% intensification of the IP staining; 3) The IP-SM method made it possible to stain semithin sections (0.5 m), and this in turn, permitted a) clear visualization under the light microscope of the intracellular distribution of antigens and, b) staining, in several adjacent sections, of roughly the same cytoplasmic region of the same cell with different primary antisera; 4) a double immunostaining whereby the first antigen in the sequence was revealed by the IP-SM method and the second by the IP procedure: 5) standardization of the IP and the IP-SM methods for post-embedding staining of ultrathin methacrylate sections.The combined application of methacrylate embedding and the IP-SM, and the use of an appropriate fixative, resulted in an ultrastructural immunocytochemical procedure characterized by a good immunoreactivity of the tissue sections, a strong and selective immunoreaction and a well preserved ultrastructure.Supported by Grant RS-82-18 from Dirección de Investigaciones, Universidad Austral de Chile, Chile     

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.     

Immunocytochemistry: its evolution and criteria for its application in the study of epon-embedded cells and tissue

The word immunocytochemistry is currently used to describe a number of methods that can be employed to localize antigens within cells by means of antigen-specific antibodies. In this article we will review a number of these methods, including immunofluorescence, immunoperoxidase, avidin-biotin, and colloidal-gold techniques. The advantages and disadvantages of the various methods are discussed, special attention being focused upon immunocytochemical staining of plastic-embedded tissue. Studies on the light microscope level show that embedding tissue in plastic prior to immunoperoxidase staining not only improves visualization of antigen-specific staining but also provides an accurate and efficient means of prescreening tissue for antigen prior to immunocytochemical staining on the electron microscope level. Varying section thickness between 1 and 3 microns does not significantly influence staining, whereas the fixative used to preserve the tissue under study does. On the electron microscope level, the colloidal gold technique appears superior to immunoperoxidase staining. It is both esthetically more pleasing and highly sensitive. Of five different colloidal gold methods tested, the most sensitive is the two-step technique that employs an antigen-specific primary antibody followed by a gold-labeled secondary antibody. Throughout this article, special emphasis is placed on the use of proper controls, both on the light and electron microscope levels. Where possible, such controls should include substitution of specific antiserum with normal serum; the use of antigen-adsorbed antiserum; the use of antisera with specificities for antigens not present in the tissue being studied; the use of tissue previously shown to be stainable for the antigen; and if cultured cells are being studied, the use of a number of cell types that do not contain the antigen.     

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.     

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.     

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.     

Immunocytochemistry of contractile and cytoskeletal proteins in smooth muscle: Lowicryl,LR White,and polyvinylalcohol compared

Summary Three embedding media have been compared with respect to post-embedding immunolabeling of contractile and cytoskeletal antigens in aldehyde-fixed smooth muscle tissue: the methacrylate derivates lowicryl K4M (cured at –35 or 60°C) and LR White (cured at 0 or 60°C) and the water soluble resin, polyvinylalcohol (dried at 60°C). Measurements of intensity of labeling of ultrathin sections in the fluorescence microscope showed that five antigens (actin, myosin light chain, tropomyosin, filamin and vinculin) reacted more or less equally with their respective antibodies in all the embedding media, including those cured at 60°C. One antibody (anti-light meromyosin) reacted well only with polyvinylalcohol-embedded tissue. In contrast to the relative invariance of antibody reactivity between media clear differences in the preservation of ultrastructural integrity were observed. Embedding in polyvinylalcohol (dried at 60°C) and in Lowicryl (cured at –35°C) resulted in superior preservation as compared to Lowicryl or LR White cured at 60°C. Examples of uitrastructural immunocytochemistry with the antibodies against filamin and myosin light chain, using the immunogold staining procedure are presented: the sites of localization by these antibodies were the same with all the media tried. The relative merits of the different methods are discussed.Abbreviations EGTA Ethyleneglycol-bis(-amino ethyl ether)N,N,N,N-tetra acetic acid - PIPES 1,4-Piperazinediethanesulfonic acid - LR London Resin     

Glycol Methacrylate in Light Microscopy a Routine Method for Embedding and Sectioning Animal Tissues

Glycol methacrylate (GMA), a water and ethanol miscible plastic, was introduced to histology as an embedding medium for electron microscopy. This medium may be made soft enough for cutting thick sections for routine light microscopy by altering its composition. A procedure for the infiltration, polymerization, and sectioning of animal tissues in GMA for light microscopy is presented which is no more complex than paraffin techniques and which has a number of advantages: (I) The GMA medium is compatible with both aqueous fixatives (formaldehyde, glutaraldehyde, Bouin's, and Zenker's) and non-aqueous fixatixes (Carnoy's, Newcomer's, ethanol, and acetone). (2) Undue solvent extraction of the tissue is avoided because adequate dehydration occurs during infiltration of the embedding medium. Separate dehydration and clearing of the tissue prior to embedding is eliminated. (3) When polymerized, the supporting matrix is firm enough that hard and soft tissues adjacent to one another may be sectioned without distortion. (4) Thermal artifact is reduced to a minimum during polymerization because the temperature of the tissue may be maintained at 0-4 C from fixation through ultraviolet light polymerization of the embedding medium. (5) Shrinkage during polymerization of the embedding medium is minimized by prepolymerization of the medium before use. (6) Sections may be easily cut using conventional steel knives and rotary microtomes at a thickness of 0.5 to 3.0 microns, thus improving resolution compared with routinely thicker paraffin sections. (7) The polymerized GMA medium is porous enough so that staining, auto radiography, and other histological procedure are done without removal of the embedding medium from the sections. A list of these stains and related procedures are included. (8) Enzyme digestion of ultra thin sections of tissue embedded in GMA is common in electron microscopic cyto chemistry. me same digestion techniques appear compatible with the thicker seaions used in light microscopy.     

Freeze-substitution and Lowicryl HM20 embedding of fixed rat brain: suitability for immunogold ultrastructural localization of neural antigens.

We examined the suitability of freeze-substitution and Lowicryl HM20 embedding of aldehyde-fixed rat brain to localize several neural antigens at the ultrastructural level. The following rabbit polyclonal and mouse monoclonal antibodies were used: affinity-purified polyclonal immunoglobulins G raised to B-50/GAP43 (a membrane-anchored, growth-associated protein); affinity-purified polyclonal immunoglobulins G to human glial fibrillary acidic protein (GFAP; a subunit of glial filaments); a polyclonal antiserum raised to adrenocorticotropic hormone[25-39] (a neuropeptide present in dense-core granules); a polyclonal antiserum raised to myelin basic protein (a protein present in compact myelin of the central nervous system); and mouse monoclonal antibodies to synaptophysin (an integral membrane protein of small synaptic vesicles). Rat mesencephalon was fixed by perfusion with buffered 2% glutaraldehyde and 4% paraformaldehyde, cryoprotected, and frozen in liquid nitrogen. Freeze-substitution of tissue was performed with anhydrous methanol and 0.5% uranyl acetate at -90 degrees C. Semi-thin Lowicryl sections were used for light microscopic visualization of B-50 in the ventromedial mesencephalic central gray substance. The procedure preserves well the ultrastructure of this region and the immunoreactivity of the selected antigens. This study shows that dehydration by freeze-substitution, combined with Lowicryl HM20 embedding at sub-zero temperature, provides a successful method of preparation of fixed brain tissue for ultrastructural studies, allowing immunogold localization of several neural antigens by double labeling in the same section and in serial sections.     

Identification of leucocyte surface antigens in paraffin- embedded bovine tissues using a modified formalin dichromate fixation

A modified fixative of formalin dichromate was combined with a cold embedding procedure for the preservation of bovine leucocyte surface antigens. Fourteen monoclonal antibodies recognizing seven bovine leucocyte surface antigens (BoCD1w2, BoCD4, BoCD8, BoWC1, BoWC3, BoWC4 and BoIgM) were applied as primary antisera in a sensitive avidin--biotin--peroxidase complex detection method. The staining results were compared with those obtained in cryostat and routinely formalin-fixed sections of corresponding tissue samples. Using the modified formalin dichromate fixative and the cold embedding procedure, all the leucocyte surface antigens tested were detectable immunohistologically in paraffin sections with a generally more distinct staining than in traditionally processed tissues. Morphological structures were better preserved than in cryostat sections but, to some extent, were poorer when compared with routinely formalin-fixed tissues. However, this method suggests that there are only mild masking effects and provides an alternative to the use of unfixed material, particularly for morphological--immunohistochemical investigations     

Embedding Plant Tissue with Plastic Using High Pressure: A New Method for Light and Electron Microscopy

An embedding technique has been developed to overcome difficulties that confront light and electron microscopists working with so-called “hard-to-embed” plant tissue. The method was originally described for freeze-dried material. It uses a modified Quickfit Rotaflo Valve and low heat to generate high pressure to aid in the infiltration and embedding of tissue with propylene oxide and plastic. The technique is not too cumbersome and requires 6 days from the dehydration step to the end of the polymerization process. Thick sections (1-2 μm) obtained from material prepared by this method stain readily with toluidine blue, and thin sections for the electron microscope stain satisfactorily following standard treatment with uranyl acetate and lead citrate. The thin sections are stable under the beam of the electron microscope. Results indicate that the quality of tissue preservation with this high pressure embedding technique is as good as that observed using standard embedding methods for electron microscopy.     

Cerium as amplifying agent--an improved cerium-perhydroxide-DAB-nickel (Ce/Ce-H2O2-DAB-Ni) method for the visualization of cerium phosphate in resin sections

A new visualization (Ce/Ce-H2O2-DAB-Ni) procedure for cerium (Ce III) phosphate in semithin and ultrathin plastic sections (Epon 812, Lowicryl K4M, glycol methacrylate) of rat kidney tissues that had been incubated before embedding for the demonstration of phosphatases (alkaline and acid phosphatase, 5(1)-nucleotidase, Mg-dependent ATPase) is described. For this purpose the hydrophobic Epon resin was removed in NaOH-ethanol solution, whereas the hydrophilic Lowicryl and methacrylate sections did not required any etching. The primary reaction product Ce III-phosphate was amplified in a Ce III-citrate solution, subsequently oxidized with H2O2 and then visualized in a H2O2 containing DAB-nickel medium (Ce IV-perhydroxy induced DAB polymerization principle). The method yielded a very clear localization of enzyme activity. The final reaction product (DAB-nickel polymers) in 0.5 - 2.0 microns semithin sections is blue-black; the background staining is completely prevented. An increase of the staining contrast was obtained by posttreatment with OsO4 (osmium black formation). Furthermore, the enzyme reaction product could be demonstrated in 40 nm thick ultrathin sections by silver intensification, which utilized the high argyrophilia of the polymerized DAB-nickel complexes. This procedure replaces the earlier published technique.     

A Method for Silver Impregnation of Mammary Myoepithelia

Histochemical methods for microscopic visualization of nummary myoepithelial cells all yielded considerable variation in completeness of myoepithelial cell staining. Although extremely variable, silver impregnation occasionally gave tissue sections containing myoepithelia having excellent microanatomical detail and contrast with other tissue elements. Consequently, sources of variation in the silver technique were considered. Composition of the tissue fixative and pH of the silver impregnating solution were most critical. A final method is presented which gives consistent, complete silver impregnation of myoepithelia, where both the cell body and cell processes are clearly evident. The staining procedure is not light sensitive, nor is acid cleaning of glassware necessary. Tissue sections from lactating mouse, rat, hamster and goat are presented; tissue from other species should stain as well. The procedure should greatly facilitate the study of the function of myoepithelial cells and the visualization of these cells in mammary pathology.     

A method for silver impregnation of mammary myoepithelia

Histochemical methods for microscopic visualization of mammary myoepithelial cells all yielded considerable variation in completeness of myoepithelial cell staining. Although extremely variable, silver impregnation occasionally gave tissue sections containing myoepithelia having excellent microanatomical detail and contrast with other tissue elements. Consequently, sources of variation in the silver technique were considered. Composition of the tissue fixative and pH of the silver impregnating solution were most critical. A final method is presented which gives consistent, complete silver impregnation of myoepithelia, where both the cell body and cell processes are clearly evident. The staining procedure is not light sensitive, nor is acid cleaning of glassware necessary. Tissue sections from lactating mouse, rat, hamster and goat are presented; tissue from other species should stain as well. The procedure should greatly facilitate the study of the function of myoepithelial cells and the visualization of these cells in mammary pathology.     

Histological investigation of organisms with hard skeletons: a case study of siliceous sponges

Siliceous and calcareous sponges commonly are treated with acid to remove the spicules prior to embedding and cutting for histological investigations. Histology of spiculated sponge tissue represents a challenging problem in sponge histotechnology. Furthermore, fluorescence in situ hybridization (FISH), a key method for studying sponge-associated microbes, is not possible after acid treatment. For a broad range of siliceous sponge species, we developed and evaluated methods for embedding in paraffin, methylmethacrylate resins, LR White resin and cryomatrix. Different methods for cutting tissue blocks as well as mounting and staining sections also were tested. Our aim was to enable histological investigations and FISH without prior removal of the spicules. To obtain an overview of tissue and skeleton arrangement, we recommend embedding tissue blocks with LR White resin combined with en bloc staining techniques for large specimens with thick and numerous spicules, but paraffin embedding and subsequent staining for whole small specimens. For FISH on siliceous sponges, we recommend Histocryl embedding if the spicule content is high, but paraffin embedding if it is low. Classical histological techniques are used for detailed tissue examinations.     

Ultrastructural visualization of concanavalin A binding sites using alkaline phosphatase-labeled Con A and ultrathin glycol methacrylate sections

Summary A method for the visualization of concanavalin A (Con A) binding sites by electron microscopy of glycol methacrylate sections is presented. This method, which is an application of the alkaline phosphatase-labeled Con A conjugate technique, solves the problems not only of limited penetration of chemicals into tissue blocks but also of injuries to tissue sections due to irritative reagents experienced in Con A-peroxidase staining. Glycol methacrylate sections are incubated successively with an alkaline phosphatase-labeled Con A solution and a lead citrate medium for the enzyme activity. Different kinds of tissues from adult rats have been used to test the method; tracheal cartilage, aorta and jejunum. The localization of Con A binding sites demonstrated by this method is consistent with the results of other published studies. Appropriate controls have been performed (ie., omission of the conjugated lectin, lectin plus its inhibitor) and these substantiate the specificity of the method.     

Pre-embedding immunohistochemistry as an approach to high resolution antigen localization at the light microscopic level

Summary Pre-embedding immunohistochemistry with subsequent embedding in hydroxypropyl methacrylate enables one to obtain high resolution staining of antigens in 1 tissue sections. A routine method using formaldehyde fixation, methanol permeation, and an indirect method with fluoresceinlabeled second antibody is described. This method is compared with other pre-embedding staining procedures. To illustrate the method the mouse small intestine was chosen as a model and stained with antibodies to tubulin, actin, and fibronectin. Some anticipated and some unusual staining patterns were found.     

Development of a simple embedding procedure allowing immunocytochemical localization at the ultrastructural level

Immunobed solution A is a water-soluble acrylic compound recently developed for immunocytochemical localization at the light microscopic level. In this study, we combined it with methyl methacrylate (MMA) to achieve sufficient hardness to obtain ultra-thin sections. Samples of platelets were dehydrated and embedded in the water-soluble acrylic mixture (WSAM). The embedding process was carried out at 4 degrees C and final polymerization was induced with either chemical (benzoyl peroxide) or physical (UV light) catalysts. Tubulin was localized at the ultrastructural level in sections embedded according to these two methods. Results were compared with those obtained in platelets processed in Lowicryl. Dehydration and embedding with the WSAM yielded a preservation of antigenicity similar to that obtained in Lowicryl. The new procedure benefits from the low temperature achieved during polymerization, providing good ultrastructural morphology and immunolocalization of protein antigens with the simplicity of a routine embedding procedure for light microscopy.     

Cytological Staining Procedures Applicable to Methacrylate-Embedded Tissues

Experiments indicate that osmic-fixed, plastic-embedded sections are suitable for examination in the light microscope. Nuclei, mitochondia, cellular membranes and cytoplasmic granules are readily demonstrable by phase microscopy. Connective tissue stains permit the identification of elastic and collagenous fibers. Glycogen and other carbohydrate-containing structures are demonstrable by the periodic acid-Schiff and the ammoniacal silver nitrate procedures. It is, therefore, possible to cross-check individual structures by comparing alternate thick and thin sections, examined in the light microscope and electron microscope respectively. Several other advantages pertain to plastic embedded tissues. The sections compare favorably in translucency and in their lack of distortion with material embedded in celloidin, yet the procedure is simpler and much more rapid. Sections of any desired thinness can be prepared, and alternate thick and thin sections are easily forthcoming. When examined in the phase-contrast microscope, mitochondrial preparations become routinely available without the uncertainties of most of the mitochondrial staining methods. It appears, therefore, that plastic embedding should find a useful place among the methods for light microscopy as well as in the armamentarium of the electron microscopist.