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.     

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.     

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

Summary 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°–50° 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.     

LR Gold embedding of nervous tissue for immunoelectron microscopy studies

Summary A major drawback of all acrylic resins commonly used for post-embedding immunocytochemical studies of the central nervous system is the disruption of the ultrastructural morphology, due to the high lipid content of neural tissue. We have investigated the suitability of the acrylic resin LR Gold, which has been employed recently for immunogold labeling studies in several non-neural tissues. Optimal preservation of both antigenicity and ultrastructure of nervous tissue was obtained after en bloc staining with uranyl acetate, followed by total dehydration in acetone and curing at low temperature. Cell membranes and myelin sheaths, which are usually lost with other acrylic resins, were well maintained. The degree of antigenicity of LR Gold-embedded tissues was comparable to that of LR White-embedded one, but the morphologic detail was much better preserved. The use of LR Gold is particularly advantageous for studying neurodegenerative disorders such as Alzheimer disease.     

LR Gold embedding of nervous tissue for immunoelectron microscopy studies.

A major drawback of all acrylic resins commonly used for post-embedding immunocytochemical studies of the central nervous system is the disruption of the ultrastructural morphology, due to the high lipid content of neural tissue. We have investigated the suitability of the acrylic resin LR Gold, which has been employed recently for immunogold labeling studies in several non-neural tissues. Optimal preservation of both antigenicity and ultrastructure of nervous tissue was obtained after en bloc staining with uranyl acetate, followed by total dehydration in acetone and curing at low temperature. Cell membranes and myelin sheaths, which are usually lost with other acrylic resins, were well maintained. The degree of antigenicity of LR Gold-embedded tissues was comparable to that of LR White-embedded one, but the morphologic detail was much better preserved. The use of LR Gold is particularly advantageous for studying neurodegenerative disorders such as Alzheimer disease.     

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.     

Immunoelectron microscopic demonstration of thyroglobulin and thyroid hormones in rat thyroid gland

We have developed a post-embedding immunogold technique for electron microscopic localization and quantitation of thyroglobulin (TG), thyroxine (T4), and triiodothyronine (T3) in rat thyroid. Labeling for TG was located on rough endoplasmic reticulum, Golgi apparatus, exocytotic vesicles, luminal colloid, colloid droplets, and lysosomes, whereas labeling for thyroid hormones was located on luminal colloid, colloid droplets, and lysosomes. We tested different procedures of fixation, dehydration, embedding, polymerization, and immunoincubation to optimize ultrastructural preservation and immunolabeling. Fixation with glutaraldehyde and osmium was possible with retained antigenicity. Dehydration temperature and the choice of embedding resin were the two crucial factors for good immunolabeling. Low-temperature dehydration greatly improved immunolabeling and could be combined with embedding in the methacrylate LR White or the epoxide Agar 100 (equivalent of Epon 812) polymerized at 40-60 degrees C, as the temperature during subsequent embedding and polymerization was of little importance for the immunoreactivity. Labeling on LR White sections was always higher than on Agar 100 sections. Various etching procedures were tested without improved specific labeling. Etching with hydrochloric acid gave nonspecific labeling of certain cell compartments.     

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.     

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.     

Ultrastructural immunogold labeling of lipid-laden enterocytes from patients with genetic malabsorption syndromes

Summary— Intestinal biopsies from patients having genetic disorders of lipoprotein assembly and secretion, such as abetalipoproteinemia (ABL) or Anderson's disease (AD), contain large amounts of lipids which are accumulated in the enterocytes. Determination of the intracellular sites in which the lipids accumulate and to which apolipoproteins the lipids are bound would help to identify the defects in these diseases and further elucidate the mechanisms by which lipoprotein assembly and secretion occur normally. Ultrastructural immunogold labeling, however, is hampered by the poor preservation of the lipids accumulated in the enterocytes of these patients. We have used routine electron microscopy (fixation and ultra-thin sectioning) along with three methods for immunogold labeling of lipid-laden enterocytes; ultrathin cryosectioning, low temperature freeze substitution with embedding in Lowicryl K4M, and ultra-low temperature freeze substitution with embedding in Lowicryl HM20, to establish a protocol for investigating the intestinal tissue from these patients. Ultracryosectioning, while preserving the overall morphology of the lipid laden enterocytes, did not preserve the lipid content and the immunogold labeling of apolipoprotein B (ApoB) appeared dislocated. Freeze substitution and low temperature embedding in Lowicryl K4M, in contrast, appeared to better preserve the lipid and lipoprotein structures; however, the antigenicity of both apoAI and apoB appeared to be lost and no specific labeling could be obtained. Freeze substitution and embedding in Lowicryl HM20 best preserved the lipid and lipoprotein structures while maintaining apoprotein antigenicity. In conclusion, immunogold labeling of apolipoproteins on lipid structures in the lipid-laden enterocytes of patients with ABL and AD is best obtained by freeze substitution and embedding in Lowicryl HM20.     

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     

Use of light microscopic immunotechniques in selecting preparation conditions and immunoprobes for ultrastructural immunolabelling of lactoferrin

Summary Successful postembedding immunolabelling for electron microscopy is sometimes difficult to achieve. We propose that light microscopy can be used (1) to detect quickly processing steps which have an adverse effect on the tissue antigenicity and (2) to check the specific reactivity of the immunogold detecting system normally employed at the ultrastructural level.The individual steps of fixation, dehydration and embedding were tested for their ability to preserve antigenicity by light microscopic peroxidase-anti-peroxidase cytochemistry. Steps that severely reduced antigenicity were replaced by less destructive alternatives compatible with reasonable ultrastructural preservation. The specific reactivity of the immunogold detecting system was assessed by using the light microscopic immunogold-silver staining method.We studied the antigen lactoferrin in human neutrophilic granulocytes from patients with chronic myeloid leukaemia. We obtained strong immunolabelling of specific granules and good ultrastructural preservation using routine methods at room temperature. For lactoferrin the method of choice was to fix in 3% paraformaldehyde/0.1% glutaraldehyde followed by 1% OsO₄, dehydrate in 70% ethanol, embed in LR White resin and polymerize at 40°C for 40h. These conditions may not be suitable for all antigens and we emphasize that for each new antigen a similar study should be carried out.     

Detection of Cell Surface Antigens in Tissue Sections by Means of Pre-Embedding Immunogold Staining

The immunogold technique has been used in electron microscopy to detect cytoplasmic and extracellular antigens by postembedding techniques. It has also been used to detect plasma-membrane-associated molecules on suspended cells and, recently, to visualise cell surface antigens in ultrathin sections of Lowicryl embedded specimens. In the present study, cell surface antigens of rat kidney and human skin were identified in tissue sections by using pre-embedding immunogold labeling. Brush border microvillar antigens and dermal lymphocyte antigens both bound numerous gold particles. The immunogold staining described here has the advantage over immunoperoxidase procedures that it is not subject to diffusion or reabsorption artifacts, and allows estimation of the antigen density on labeled cells. Furthermore, this pre-embedding immunogold technique is ideally suited to detecting cell surface-associated antigens since it preserves antigenicity, allows gold particle penetration and enhances cell membrane profiles.     

Detection of cell surface antigens in tissue sections by means of pre-embedding immunogold staining

The immunogold technique has been used in electron microscopy to detect cytoplasmic and extracellular antigens by postembedding techniques. It has also been used to detect plasma-membrane-associated molecules on suspended cells and, recently, to visualize cell surface antigens in ultrathin sections of Lowicryl embedded specimens. In the present study, cell surface antigens of rat kidney and human skin were identified in tissue sections by using pre-embedding immunogold labeling. Brush border microvillar antigens and dermal lymphocyte antigens both bound numerous gold particles. The immunogold staining described here has the advantage over immunoperoxidase procedures that is not subject to diffusion or reabsorption artifacts, and allows estimation of the antigen density on labeled cells. Furthermore, this pre-embedding immunogold technique is ideally suited to detecting cell surface-associated antigens since it preserves antigenicity, allows gold particle penetration and enhances cell membrane profiles.     

A method for using low-temperature embedding media for electron microscopy of cells grown on microporous supports.

Attempts were made to embed Madin-Darby canine kidney (MDCK) cells grown on nitrocellulose microporous supports in Lowicryl, a medium designed for the retention of antigenicity for electron microscopic immunocytochemical studies. It was found that the membrane fragmented during the prescribed embedding procedure leading to loss of the cell sample. This necessitated the formulation of a new combination of fixative and dehydration agents which would allow: (i) preservation of membrane and cellular integrity; (ii) infiltration and embedding in Lowicryl; and (iii) detection of a specific antigen in thin sections. The cellular monolayer and organelle profiles were best preserved with glutaraldehyde fixation at room temperature followed by ethanol dehydration. Since the latter was carried out at temperatures attainable with an ice-salt bath, there was no need for a special ultralow-temperature apparatus. This procedure was applied to a MDCK cell clone that consisted of stable secretors of human growth hormone (hGH), as a result of being transfected with a plasmid containing the hGH gene. Thus, it was demonstrated that hGH could be detected by immunogold labeling of thin sections and localized to specific cellular structures. The procedure developed in this report is applicable to cells grown on two other supports and may be extended further.     

Post embedding immunoelectron microscopy of human breast cancer: a comparison of three acrylic resins

Summary The suitability of three acrylic resins for the immunoelectron microscopical localization of cell surface and cytoskeletal antigens in surgically excised, immersion fixed human breast cancer, using an immunogold system, has been assessed.Good localization of milk fat globule membrane was achieved with LR White, LR Gold and Lowicryl K11M, although the embedding schedule for LR White had to be modified. The best results were achieved with Lowicryl K11M. Only scanty labelling of actin and cytokeratin was seen in LR White embedded tissue, whereas there was clear localization in LR Gold and Lowicryl K11M embedded samples. Tubulin and -actinin was detected at low level in tissues in the low temperature embedding resins, but not in LR White embedded samples. The morphology of the latter was poorer, and there was greater variability in ultrastructure and labelling.Of the two low temperature embedding resins, Lowicryl K11M gave slightly better results. However, the advantages could be outweighed by the problem incurred in achieving the low temperatures, and by poorer handling properties than LR Gold.     

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.     

Ultrastructural detection of DNA-incorporated bromodeoxyuridine in resin embedded brain tissue

We describe a protocol for the ultrastructural detection of DNA-incorporated bromodeoxyuridine (BUdR) in resin embedded tissue by means of post-embedding immunogold labeling. The paraventricular zone of rat embryos brains was dissected, fixed either in paraformaldehyde or glutaraldehyde, and embedded in LR White. BUdR gold labeling was only found when thin sections were pretreated with 4 N HCl. Other DNA denaturing agents, such as Na ethoxide, formamide, formic acid, heat or HCl at lower concentrations were ineffective. Very little difference in the degree of labeling was found depending on the fixation. This method can be applied to investigate the fine structure of replicating cells in other in vivo conditions, such as human tumors.     

Ultrastructural immunolocalization of polyamines in HeLa cells subjected to fast-freezing fixation and freeze substitution

 Polyamines have been localized at the ultrastructural level in HeLa cells subjected first to fast-freezing fixation (FFF)-freeze substitution (FS) and then to an immunocytochemical method combining anti-polyamine antibodies and immunogold labelling. Polyamines were found in both the cytoplasm and the nucleus and, in the latter, particularly over the dense chromatin area. To our knowledge, this is the first example of the electron microscopic localization of a hapten after FFF-FS. For the preservation of fine ultrastructural details, this FFF-FS method is not only adequate but also greatly reduces, if not totally eliminates, any leeching-out and redistribution of the polyamines during the preparation of the sample. For the preservation of antigenicity in situ during FS, epoxy resin was more effective than hydrophilic LR white resin, probably due to the solubility of polyamines in the latter. Accepted: 12 November 1996     

Ultrastructural detection of DNA-incorporated bromodeoxyuridine in resin embedded brain tissue

Summary We describe a protocol for the ultrastructural detection of DNA-incorporated bromodeoxyuridine (BUdR) in resin embedded tissue by means of post-embedding immunogold labeling. The paraventricular zone of rat embryos brains was dissected, fixed either in paraformaldehyde or glutaraldehyde, and embedded in LR White. BUdR gold labeling was only found when thin sections were pretreated with 4 N HCl. Other DNA denaturing agents, such as Na ethoxide, formamide, formic acid, heat or HCl at lower concentrations were ineffective. Very little difference in the degree of labeling was found depending on the fixation. This method can be applied to investigate the fine structure of replicating cells in other in vivo conditions, such as human tumors.