Immunogold-silver staining of mesangial antigen in lowicryl K4M- and LR gold-embedded renal tissue using epipolarization microscopy.

We examined the localization of a glomerular mesangial antigen with a Thy-1.1 monoclonal antibody by epipolarization microscopy (EPI) of silver-enhanced, immunogold-stained renal tissue embedded in LR Gold and Lowicryl K4M, and compared the attributes of these hydrophilic resins. Antigen was well preserved in tissue embedded in both resins. LR Gold-embedded tissue demonstrated excellent immunostaining properties, sectioned more easily, and showed better durability during staining than K4M. Lowicryl K4M-embedded tissue, however, displayed a phenomenon of self-illumination when counterstained with eosin which was not seen with LR Gold. This enabled immunostaining to be precisely related to tissue morphology without the necessity of simultaneous transillumination, which can be problematic when used in combination with EPI because of reflection of incident illumination from sub-stage optical surfaces.     

Influence of embedding media in prolactin labelling with immunogold techniques

Summary Immunogold labelling of prolactin in three different embedding media was compared. The polymeric prolactin in secretory granules was labelled in the three media, however, acrylic monomers (Lowicryl K4M and LR White) provided a more intense labelling with higher dilutions of the primary antibody, compared to the labelling in the epoxy resin (araldite). An intense labelling of monomeric prolactin in Golgi complex was detected only in acrylic embedments, and the labelling on the rough endoplasmic reticulum was significant only in LR White embedded tissues.     

Immunoelectron microscopical localization of ornithine transcarbamylase in hepatic parenchymal cells of the rat

Summary The electron microscopical localization of ornithine transcarbamylase in rat liver was investigated by a protein A—gold technique applied to thin sections of Lowicryl K4M- or LR gold-embedded materials and to ultracryosections. Gold particles were exclusively confined to mitochondria of the parenchymal cells but not of sinus-lining cells. In mitochondria, gold particles were present in the matrix and closely associated with the inner membrane. The most intensive labelling was obtained from ultracryosections, while weaker labelling was noted in sections of materials embedded in both Lowicryl K4M and LR gold. The association of the enzyme with the inner membrane was confirmed by quantitative analysis of distribution pattern.     

Ultrastructural and nuclear antigen preservation after high-pressure freezing/freeze-substitution and low-temperature LR White embedding of HeLa cells

A protocol for high-pressure freezing and LR White embedding of mammalian cells suitable for fine ultrastructural studies in combination with immunogold labelling is presented. HeLa S3 cells enclosed in low-temperature gelling agarose were high-pressure frozen, freeze-substituted in acetone, and embedded in LR White at 0°C. The morphology of such cells and the preservation of nuclear antigens were excellent in comparison with chemically fixed cells embedded in the same resin. The immunolabelling signal for different nuclear antigens was 4-to-13 times higher in high-pressure frozen than in chemically fixed cells. We conclude that one can successfully use high-pressure freezing/freeze-substitution and LR White embedding as an alternative of Lowicryl resins.     

Immunocytochemical demonstration of serine:pyruvate aminotransferase in peroxisomes and mitochondria of rat kidney

Summary The light- and electron-microscopic localization of serine:pyruvate aminotransferase (SPT) in rat kidney was studied using immunoenzyme and protein A-gold techniques. Rat kidneys were fixed by perfusion through the abdominal aorta and small tissue slices were embedded in Epon, Lowicryl K4M, or LR Gold. The Epon was removed from the semithin sections, which were then stained using the immunoenzyme technique. Ultrathin sections of Lowicryl K4M- or LR gold-embedded materials were labeled using the protein A-gold technique. At light microscopy, discrete granular reaction deposits were exclusively present in the proximal tubule, all of whose segments were positive for SPT. A weakly positive reaction was observed in the distal tubules. At electron microscopy, gold particles indicating the antigenic sites for SPT were confined to the peroxisomes and mitochondria. The labeling intensity of both organelles was dependent on the embedding resins used. The labeling of Lowicryl K4M-embedded material was weaker than that of LR gold-embedded material; Quantitative analysis confirmed this result. Our results indicate that, in rat kidney, the main intracellular sites for SPT are peroxisomes and mitochondria of the proximal tubule.     

Immunocytochemical demonstration of serine: pyruvate amino-transferase in peroxisomes and mitochondria of rat kidney

The light- and electron-microscopic localization of serine: pyruvate aminotransferase (SPT) in rat kidney was studied using immunoenzyme and protein A-gold techniques. Rat kidneys were fixed by perfusion through the abdominal aorta and small tissue slices were embedded in Epon, Lowicryl K4M, or LR Gold. The Epon was removed from the semithin sections, which were then stained using the immunoenzyme technique. Ultrathin sections of Lowicryl K4M- or LR gold-embedded materials were labeled using the protein A-gold technique. At light microscopy, discrete granular reaction deposits were exclusively present in the proximal tubule, all of whose segments were positive for SPT. A weakly positive reaction was observed in the distal tubules. At electron microscopy, gold particles indicating the antigenic sites for SPT were confined to the peroxisomes and mitochondria. The labeling intensity of both organelles was dependent on the embedding resins used. The labeling of Lowicryl K4M-embedded material was weaker than that of LR gold-embedded material; Quantitative analysis confirmed this result. Our results indicate that, in rat kidney, the main intracellular sites for SPT are peroxisomes and mitochondria of the proximal tubule.     

Demonstration of anionic sites on the luminal and abluminal fronts of endothelial cells with poly-L-lysine-gold complex

An attempt was made to demonstrate the anionic sites on the endothelial cell (EC) surfaces of mouse brain micro-blood vessels (MBVs) after embedding of tissue samples in hydrophilic media: Lowicryl K4M, LR White, and Polyamph-10. As a cationic probe, poly-L-lysine-gold complex (PLG), prepared according to the procedure of Skutelsky and Roth (J Histochem Cytochem 34:693, 1986), was used. In ultra-thin sections of brain samples embedded in Lowicryl K4M and LR White, the anionic sites were demonstrated in the entire cross-section of the vessel wall. After embedding in Polyamph-10, however, the anionic sites could not be detected. Brain capillaries, representing blood-brain barrier type MBVs, showed polar distribution of anionic sites, evidenced by more intense labeling of luminal than of abluminal plasma membrane of the EC. Some differences in labeling of ECs and of basement membrane in arterioles and venules were also noted. The use of cationic gold and the ultra-thin sections of tissue samples embedded in hydrophilic media (Lowicryl K4M and LR White) seems to be a promising new method for detection of anionic constituents located on both luminal and abluminal surfaces of the EC, in the basement membrane, and in other components of the vessel wall.     

Immunogold localization of TGFβ 1 protein and mRNA in human skin using a colloidal gold/digoxygenin system

Tissue preservation, and immunogold cytochemical and in-situ hybridization labelling intensities vary according to the preparatory protocols used. We wished to determine which preparative protocols produce optimal preservation, protein and mRNA labelling. Nine combinations of fixative and embedding resin were therefore studied using postembedding immunoelectron microscopy and a novel immunogold digoxygenin in situ hybridization (ISH) system, to quantitate the presence of transforming growth factor beta 1 (TGF 1) protein and message in human skin. The best preservation was observed in tissue fixed in 1% glutaraldehyde and embedded in LR White resin or low acid glycolmethacrylate resin (LA-GMA). Preservation was poor in tissue fixed with 1% glutaraldehyde and fair in 4% paraformaldeyde, when embedded in Unicryl. Ethanediol dehydration coupled with LA-GMA embedding resulted in reasonable preservation. Based on quantitative measures of the labelling density for TGF 1 protein and mRNA, immunogold labelling was adequate with 1% glutaraldehyde fixation coupled with LR White or LA-GMA resins, and also with 4% paraformaldehyde and LR White resin, but was best with ethanediol dehydration and LA-GMA embedding. ISH labelling under basal conditions was best in LA-GMA with 1% glutaraldehyde or 4% paraformaldehyde. The ISH label in tissue fixed with 1% glutaraldehyde and embedded in LA-GMA was significantly increased by treatment with proteinase K. Overall, ethanediol dehydration was associated with a good immunoelectron microscopic (IEM) label while LA-GMA with 1% glutaraldehyde or 4% paraformaldehyde resulted in a consistently detectable ISH label. LA-GMA embedding with 1% glutaraldehyde fixation gave a good result with both IEM and ISH labelling.     

Detection of p24 in HIV-1 infected cells embedded in LR White and Lowicryl K4M

Summary In this study we present a postembedding on-grid immunogold labelling procedure for the ultrastructural localization of the HIV-1 core protein p24. HIV-1 infected cells were fixed in 0.1% glutaraldehyde, incompletely dehydrated and embedded in LR White or in Lowicryl K4M. Antigenic sites were detected by incubation of ultrathin sections with primary mouse monoclonal antibody anti-HIV-1 p24, followed by the secondary antibody goat anti-mouse IgG coupled to 10nm gold particles. Antigenicity of p24 was found to withstand the applied fixation and was shown to be preserved in LR White as well as in Lowicryl. The described procedure permits the uncomplicated and easy detection of p24 in HIV-1 infected cells and tissues.     

Immunodetection of low concentrations of ovalbumin in cryoprocessed quail oviduct cells by semi-automatic quantitative analysis

Summary— In a previous paper (Quintana et al, Biol Cell 72, (1991) 167–180) we reported the anti-ovalbumin antibody immunolabeling in the cytoplasm of tubular gland cells from cryofixed, cryosubstituted and acrylic-resin embedded quail oviduct. To confirm these preliminary visual observations, we have carried out a semi-automatic quantitative study of immunogold labeling. The quantitative results confirm the previous data. In addition, we found a significantly higher immunolabeling with low temperature Lowicryl K11M embedding procedure as compared with high temperature LR White embedding.     

Applicability of using acrylic resins in post-embedding ultrastructural immunolabelling of human neutrophil granule proteins

Summary In this study, quantitative assessments were carried out, (1) by light microscopy during tissue preparation for electron microscopy and (2) by electron microscopy after on-grid immunogold staining, to determine the suitability of using LR White and Lowicryl K4M thin sections to identify lactoferrin and elastase in the granules of human neutrophil leucocytes. Quantitative assessment of the effect of fixation, dehydration and embedding on the preservation of antigenicity during tissue preparation for electron microscopy, using light microscopic peroxidase anti-peroxidase immunocytochemistry, enabled the selection of preparation conditions that adequately preserved both antigenicity and ultrastructure. OsO₄ post-fixation, following primary aldehyde fixation, improved the retention of antigenicity during dehydration and embedding and the preservation of fine structure. Partial rather than complete dehydration retained more of the antigenicity. The efficiency, sensitivity and resolution of immunolabelling and the ultrastructure and quality of sections achieved after embedding in LR White were superior to those obtained after embedding in Lowicryl K4M. Consequently room temperature embedding in LR White following double fixation and partial dehydration is a better and more reliable preparation technique than low-temperature embedding in Lowicryl K4M following single fixation and partial dehydration for localizing lactoferrin and elastase to the specific and primary granules respectively in human neutrophilic granulocytes by the on-grid immunogold staining method.     

Effects of monomeric acrylic embedding media on the antigenicity of two epitopes of the MIC2-encoded Ewing's sarcoma cell membrane antigen.

Comparative electron microscopic studies of pre- and postembedding immunolabeling experiments have shown that the antigenicity of some epitopes is lost during acrylic resin embedding of the respective tissues. In the present investigation we have tested the sensitivities of two embedding-labile epitopes (HBA-71 and HBA-45) of the Ewing's sarcoma-associated MIC2-encoded E2 antigen to the effects of the different treatment steps, which are necessary for the preparation of ultrathin sections. The extent of antigenic retention was quantitated using flow cytometry and enzyme-linked immunosorbent assays (ELISA) of tumor cell lines, thymocytes and cell membrane extracts. Fixation, dehydration and high temperature treatment of MIC2-positive cells showed only minor effects on the reactivity with the HBA-71 and HBA-45 antibodies. However, exposure of the cells to the monomeric acrylic resins LR White (LRW), LR Gold (LRG) and Lowicryl K4M at 4 degrees C for 2-18 h resulted in a significant reduction of the HBA-71 and HBA-45 reactivities. In contrast, the antigenicity of both epitopes was maintained during treatment with the apolar Lowicryl HM20 embedding medium under these non-polymerizing conditions. The resins have no direct effect on the HBA-71/HBA-45 antigen, since it could be extracted in intact form from membranes of native, but not of fixed, tumour cells using LRW for membrane solubilization. These data indicate that the HBA-71/HBA-45 antigen remains in the cell membrane and is indirectly influenced by the extraction/modification of adjacent membrane constituents. The adverse effects of the polymerization process, in the case of embedding at low temperature in Lowicryl HM20, destroyed MIC2-antigenicity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of monomeric acrylic embedding media on the antigenicity of two epitopes of the MIC2-encoded Ewing's sarcoma cell membrane antigen

Summary Comparative electron microscopic studies of pre- and postembedding immunolabeling experiments have shown that the antigenicity of some epitopes is lost during acrylic resin embedding of the respective tissues. In the present investigation we have tested the sensitivities of two embedding-labile epitopes (HBA-71 and HBA-45) of the Ewing's sarcoma-associated MIC2-encoded E2 antigen to the effects of the different treatment steps, which are necessary for the preparation of ultrathin sections. The extent of antigenic retention was quantitated using flow cytometry and enzyme-linked immunosorbent assays (ELISA) of tumor cell lines, thymocytes and cell membrane extracts. Fixation, dehydration and high temperature treatment of MIC2-positive cells showed only minor effects on the reactivity with the HBA-71 and HBA-45 antibodies. However, exposure of the cells to the monomeric acrylic resins LR White (LRW), LR Gold (LRG) and Lowicryl K4M at 4° C for 2–18 h resulted in a significant reduction of the HBA-71 and HBA-45 reactivities. In contrast, the antigenicity of both epitopes was maintained during treatment with the apolar Lowicryl HM20 embedding medium under these non-polymerizing conditions. The resins have no direct effect on the HBA-71/HBA-45 antigen, since it could be extracted in intact form from membranes of native, but not of fixed, tumour cells using LRW for membrane solubilization. These data indicate that the HBA-71/HBA-45 antigen remains in the cell membrane and is indirectly influenced by the extraction/modification of adjacent membrane constituents. The adverse effects of the polymerization process, in the case of embedding at low temperature in Lowicryl HM20, destroyed MIC2-antigenicity. In addition, the changes in tissue antigens induced by monomeric resins seem to be an important primary source of negative results in postembedding immunolabeling of integral glycosylated cell surface proteins by antibodies and lectins.     

Lectin cytochemistry in the gastrointestinal tract with special reference to glycosylation in the Golgi apparatus of Brunner's gland cells.

Two hydrophilic, low temperature-embedding resins, Lowicryl K4M and LR White, were compared in lectin cytochemistry. Post-embedding staining of colloidal gold-labeled Griffonia symplicifolia agglutinin II (GSA-II) resulted in staining of the Golgi apparatus and mucous granules of mucous neck cells in the gastric fundic gland, pylorocytes, and Brunner's gland cells embedded in either resin, although it was much easier to make ultra-thin sections with LR White-embedded material than with the other. Post-fixation with uranyl acetate followed by LR White embedding improved general ultrastructure so that lectin binding sites were identified precisely. All examined lectins, soybean agglutinin (SBA), Maclura pomifera agglutinin (MPA), GSA-II, and Ulex europaeus agglutinin I (UEA-I), stained mucous granules and the Golgi apparatus, in which the staining pattern was characteristic of each lectin: cis cisternae were labeled with SBA and MPA, intermediate cisternae with GSA-II, and trans cisternae and mucous granules with SBA, GSA-II, UEA-I, and lightly with MPA. No labeling was observed in the rough endoplasmic reticulum with any lectin. These findings suggest that the Golgi apparatus is the site of O-linked glycosylation and can be divided into at least three distinct compartments with regard to the glycosylation.     

Fine localization of serine: pyruvate aminotransferase in rat hepatocytes revealed by a post-embedding immunocytochemical technique

Summary Immunocytochemical localization of serine: pyruvate aminotransferase (SPT) in rat hepatocytes was studied using a protien A-gold technique. Rat liver was fixed by perfusion. Vibratome sections (100 m thick) of the liver were embedded in Epon or Lowicryl K4M. Ultrathin sections were incubated with antiSPT, followed by protein A-gold complex. Gold particles representing the antigenic sites for SPT were seen in three subcellular compartments, peroxisomes, mitochondria, and cytoplasm. In the control experiments the specificity of the immunolabelling was confirmed. Quantitative analysis of the labelling density showed that main subcellular compartments containing SPT are mitochondria and peroxisomes. In addition, the gold particles distributing in the cytoplasm were 16%–29% of the total labelling. The result indicated that the cytoplasm also contains SPT with a low density.     

Post-embedding methods for immunolocalization of elastin and related components in tissues

Elastic tissue is composed of amorphous-appearing elastin and 12-nm diameter microfibrils, one component of which has recently been isolated and characterized as the 31 KD microfibril-associated glycoprotein MAGP. Monospecific antibodies to each of these components have been developed in this laboratory. The parameters that determine optimal localization of colloidal gold probes for post-embedding immunolabeling of elastic tissue components have been systematically studied in a variety of normal and developing tissues in mammals and birds. Protein A-gold probes stabilized with dextran have been shown to provide complexes that remain stable after more than 2 years. Conditions have been defined that permit precise localization within the extracellular matrix of antibodies to MAGP and to elastin, singly and together. Best results were obtained with acrylic resins (Lowicryl K4M or LR White). Fixation in glutaraldehyde or other aldehydic fixatives, with or without osmium, did not affect the immunostaining of elastic tissue with affinity-purified antibodies to tropoelastin, or to anti-[alpha-elastin] or anti-[alkali-insoluble elastin]. Immunostaining with the anti-MAGP antibody was less robust and was possible in tissues which had been fixed only lightly before embedding in Lowicryl K4M or LR White. This staining was enhanced by metaperiodate oxidation of the sections as well as by reduction of the tissues with sodium borohydride en bloc, followed by hyaluronidase digestion of the sections. The effects on immunostaining of a range of enzyme digestions have also been examined. Conditions have thus been defined that make possible detailed study of the relationship between elastic tissue, elastin-associated microfibrils, and other microfibrillar structures in normal and abnormal tissues during development and aging.     

Comparison of LR white resin, Lowicryl K4M and Epon postembedding procedures for immunogold staining of actin in the testis

The efficiency of various postembedding procedures for actin immunogold detection was compared using testicular tissue as a model. Whatever the fixative, testes embedded in LR White resin or in Lowicryl K4M showed few differences as regard ultrastructural preservation and gave similar actin antigenicity preservation. A purified polyclonal antibody (IgG) and a monoclonal antibody (IgM) visualized with gold secondary antibody yielded high labeling intensity whereas the IgG-protein-A gold association was less efficient. Crude antisera gave a low specific staining/background ratio. Samples of testes, fixed in different conditions, were also embedded in Epon, omitting propylene oxide and lowering polymerization temperature to 40 degrees-50 degrees C. This slight modification improved ultrastructural preservation which was better than with hydrophilic resins, as well as made possible immunogold detection of actin though antigenicity preservation was lesser than with these resins. Thus, in Epon embedded samples actin labeling, using IgG antiactin-gold secondary antibody, was similar to that observed after hydrophilic resin-protein-A gold procedures. In addition to actin labeling of various somatic cells it was confirmed that actin is a consistent component of the subacrosomal space of spermatids during the greater part of spermiogenesis in rat.     

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     

Ultrastructural immunocytochemistry of glia cells. Double labeling studies using LR White embedding and colloidal gold

The introduction of acrylate resins (Lowicryl K4M, LR White) into electronmicroscopic immunocytochemistry applied to embedded tissue (post-embedding method) has improved the localization of antigens because of a satisfactory preservation of both ultrastructure and antigenicity of tissues. Here we describe a method that allows double staining of intracellular and membranous determinants in ultrathin sections of nervous tissue and cultures of peripheral nervous system cells. Ultrathin sections of the rat central nervous system fixed on uncoated grids were stained first for MBP selectively on the one face, then the opposite face was stained for GFAP using monoclonal antibodies and indirect immunogold staining method (IGS). Cultured Schwann cells induced to express major histocompatibility complex (MHC) class II antigens were stained for class II antigens by pre-embedding method then followed by post-embedding IGS for the other intracytoplasmic antigens.     

Flavonol ring B-specific O-glucosyltransferases: purification, production of polyclonal antibodies, and immunolocalization.

UDP-glucose: flavonol 2'- and 5'-O-glucosyltransferases (E.C.2.4.1.-) from leaves of Chrysosplenium americanum were copurified to apparent homogeneity by successive chromatography on Sephacryl S-200, UDP-glucuronic acid-agarose, Mono P, Superose 12, and Mono Q columns. Both enzymes have similar properties except for their substrate specificity and stability (J. Chromatogr. 388, 235, 1987). The purified protein was used as the source of antigen to produce polyclonal antibodies in rabbits. In situ localization of the O-glucosyltransferases was studied by applying a postembedding immunogold labeling technique on ultrathin sections of Lowicryl K4M- and LR White-embedded tissues. Postfixation with osmium tetroxide followed by embedding in LR White resulted in good preservation of membrane ultrastructure, although protein antigenicity was greatly reduced. Leaf sections embedded in Lowicryl K4M had an extracted appearance; however, they retained a high degree of protein antigenicity revealing the deposition of gold particles in the periplasmic region of cells. Considering the compromise chosen in this study to retain antigenicity over preservation of membrane ultrastructure, the results suggest that the "easily solubilized" O-glucosyltransferases of C. americanum may actually be associated with vesicle-like structures and cytoplasmic membranes.