Optimization of immunogold labeling TEM: an ELISA-based method for evaluation of blocking agents for quantitative detection of antigen.

We developed an ELISA-based method for rapid selection of optimal blocking agents to be used in antigen quantification by immunogold labeling electron microscopy. Casein, skim milk, BSA from two sources, acetylated BSA, fish skin gelatin, horse serum, and goat serum were tested for their ability to block nonspecific binding of antibody to recombinant Vitreoscilla hemoglobin (VHb) antigen expressed in Escherichia coli cells by ELISA and the results were confirmed by quantitative immunogold labeling transmission electron microscopy (TEM). Ability to minimize NSB was also evaluated by dot-blot and Western blotting methods. The results demonstrated that ELISA was most accurate in predicting the most efficient blocking agent for TEM. Existing methods could not provide an accurate picture of the ability of various reagents to suppress background labeling. The sensitivity of detection of antigens by immunoelectron microscopy depends on the assay procedure being optimized to obtain the highest possible signal along with as low a background (noise) as possible. Our study indicated that an ELISA-based evaluation of various blocking agents could help in the rapid selection and optimization of a suitable protocol for immunogold localization and quantification of antigens by TEM.     

Improved Procedures for Electron Microscopic Visualization of the Cytoskeleton of Cultured Cells

We have developed an improved electron microscopic procedure appropriate for correlative light and electron microscopy of the cytoskeleton. The procedure is based on detergent extraction, chemical fixation, critical point drying, and platinum/carbon coating of cultured cells and the improvements consist of modifications which are minor individually but collectively of substantial impact. They are: inclusion of polyethylene glycol into the extraction medium; cell lysis at room temperature; fixation by sequential application of glutaraldehyde, tannic acid, and uranyl acetate; horizontal position of specimens during dehydration and drying; and uranyl acetate treatment during dehydration. As a result, we have obtained a greatly improved quality of electron microscopic images together with a high consistency of results. Long and straight actin filaments were clearly seen in stress fibers and newly formed lamellipodia. Their polarity was distinctly revealed by decoration with myosin subfragment 1. Depletion of actin from cytoskeletons by gelsolin treatment allowed for better visualization of myosin, intermediate filaments, and microtubules. Intermediate filaments exposed by this treatment exhibited numerous side projections in a hitherto unreported millipede-like appearance. The suggested procedure was compatible with immunogold labeling as demonstrated with an antibody to tubulin. Correlative light and electron microscopy of cells microinjected with a fluorescent derivative of myosin II was reliable and efficient, producing a close resemblance between the two kinds of images.     

Application of microwave technology to the processing and immunolabeling of plastic-embedded and cryosections.

We have adapted existing microwave irradiation (MWI) protocols and applied them to the processing and immunoelectron microscopy of both plastic-embedded and frozen sections. Rat livers were fixed by rapid MW irradiation in a mild fixation solution. Fixed liver tissue was either cryosectioned or dehydrated and embedded in Spurr's, Unicryl, or LR White resin. Frozen sections and sections of acrylic-embedded tissue were immunolabeled in the MW oven with an anti-catalase antibody, followed by gold labeling. Controls were processed conventionally at room temperature (RT). The use of MWI greatly shortened the fixation, processing, and immunolabeling times without compromising the quality of ultrastructural preservation and the specificity of labeling. The higher immunogold labeling intensity was achieved after a 15-min incubation of primary antibody and gold markers under discontinued MWI at 37C. Quantification of the immunolabeling for catalase indicated a density increase of up to fourfold in the sections immunolabeled in the MW oven over that of samples immunolabeled at RT. These studies define the general conditions of fixation and immunolabeling for both acrylic resin-embedded material and frozen sections.     

An artificial test substrate for evaluating electron microscopic immunocytochemical labeling reactions

We describe an artificial substrate system for optimization of labeling parameters in electron microscope immunocytochemical studies. The system involves use of blocks of glutaraldehyde-polymerized BSA into which a desired antigen is incorporated by a simple soaking procedure. The resulting antigen-impregnated artificial substrate can then be fixed and embedded identically to a piece of tissue. The BSA substrate can also be dried and then sectioned for immunolabeling with or without chemical fixation and without exposing the antigen to dehydrating agents and embedding resins. The effects of various fixation and embedding procedures can thus be evaluated separately. Other parameters affecting immunocytochemical labeling, such as antibody and conjugate concentration, can also be evaluated. We used this system, along with immunogold labeling, to determine quantitatively the optimal fixation and embedding conditions for labeling of hepatitis B surface antigen (HbsAg), human IgG, and horseradish peroxidase. Using unfixed and unembedded HBsAg, we were able to detect antigen concentrations below 20 micrograms/ml. We have shown that it is not possible to label HBsAg within resin-embedded cells using conventional aldehyde fixation protocols and polyclonal antibodies.     

Post-embedding double-labeling of antigen-retrieved ultrathin sections using a silver enhancement-controlled sequential immunogold (SECSI) technique.

In electron microscopy, the post-embedding immunogold technique provides a high degree of resolution and the possibility of quantitation owing to the intrinsic characteristics of the colloidal gold marker. Application of this technique to the subcellular localization of multiple antigens by differential labeling using gold markers of different sizes, or to double labeling using the same primary antibody isotype with serial silver enhancement, has been reported. We have incorporated this double labeling technique into a modified procedure that produces excellent labeling and ultrastructural preservation, even after exposure of ultrathin sections large enough to cover a 300- micro m-diameter single-hole grid to hot antigen retrieval solutions and prolonged labeling protocols.     

Evidence for perforin-like activity associated with earthworm leukocytes

Earthworm (Eisenia fetida) coelomic fluid contains several leukocytes (coelomocytes): basophils, acidophils and neutrophils as well as chloragocytes. Small coelomocytes and coelomocyte lysate are cytotoxic for the tumor cell target K562. The expression of a lytic factor was investigated by immunocytochemistry using light and transmission electron microscopy. A rat-anti-mouse-perforin-mAb labeled mainly small coelomocytes (nearly 20%) as visualized by light microscopy. TEM analysis using immunogold showed a homogenous labeling in the cytoplasm of small coelomocytes. The highest number of immunogold particles was estimated in coelomocytes with many small cytoplasmic granules. Coelomocytes with large lysosomal granules were also labeled but less intensely. No antibody binding was observed for chloragocytes either in light or electron microscopy. This suggests that the perforin-like activity is associated with only one cell type and that chloragocytes are responsible for other lytic activities. MALDI-MS revealed calreticulin usually associated with perforin in mammalian cells that mediate lysis (e.g. NK, CTL). Together, results strongly suggest the presence of putative perforin in earthworms. This in turn supports the hypothesis that perforin is a conserved component important in immune defense during evolution.     

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.     

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.     

Antigen retrieval trial for post-embedding immunoelectron microscopy by heating with several unmasking solutions.

A novel antigen retrieval procedure was carried out in the post-embedding immunogold electron microscopy method to improve the stainability of the samples. This was done by weakly fixing cultured Helicobacter pylori (ATCC43504) and embedding in Lowicryl K4M. Before staining with the anti-H. pylori antibody, the ultrathin sections were mounted on a nickel grid and heated at 121C for 15 min, 99C for 40 min, and 65C for 24 hr in distilled water, 0.1 M phosphate buffer (pH 7.4), 0.01 M EDTA (pH 7.2), 0.05 M Tris buffer (pH 10.0), 0.8 M urea (pH 7.2), 0.01 M citric acid (pH 6.0), or a commercially available target unmasking fluid (S1699; pH 6.0). Antigen retrieval in the Tris buffer solution generally showed better stainability than the classical post-embedding method without any antigen retrieval. At 65C for 24 hr, better stainability of the ultrasections was observed for each of the solutions used except for the phosphate buffer compared to the control. We suggest that the antigen retrieval method should be applied for routine use even by in post-embedding immunogold electron microscopy.     

Re-evaluation of epoxy resin sections for light and electron microscopic immunostaining.

Epoxy resins provide optimal tissue morphology at both the light and the electron microscopic level and therefore enable correlative studies on semithin and thin sections from the same tissue block. Here we report on an approach to retain these advantages for immunolabeling studies by adapting and combining well-known techniques, i.e., surface etching with sodium ethoxide and heat-mediated antigen retrieval. We propose a simple procedure for immunostaining semithin and thin epoxy resin sections. To check its applicability, well characterized, commercially available antibodies (against E-cadherin, alpha-catenin, and beta-catenin) were used on sections of human small intestine. By light microscopy, the immunostaining efficiency was compared on cryo-, paraffin, and epoxy semithin sections processed in parallel. The most detailed results were obtained on semithin sections, where the labeling precisely delineated the lateral plasma membrane of the enterocytes. At the electron microscopic level the procedure did not damage the structures and allowed an efficient, reproducible immunogold labeling extending homogeneously over exceptionally wide tissue areas. The three antibodies specifically labeled the zonula adherens of the junctional complex between epithelial cells and, in agreement with light microscopic observations, the lateral plasma membrane.     

Applications of immunogold and lectin-gold labeling in tumor research and diagnosis

Immunohistochemistry and carbohydrate histochemistry have had an enormous impact on both tumor research and diagnosis. In particular, immunogold labeling has provided significant advantages over classical fluorescence and enzyme-based techniques. In light microscopy, the silver-intensified gold labeling has proven highly sensitive and precise in localization. In electron microscopy, the gold particle marker was a prerequisite for succesful and unequivocal antigen detection in electron-dense cellular structures such as secretory granules. In this review we demonstrate the usefulness of light and electron microscopiaal gold labeling techniques as applied in tumor research and diagnosis. The examples include expression of β-1,6 branches and specific sialoglycoconjugates in colon carcinoma, b-12 carbohydrate epitope in breast carcinoma, polysialic acid in neuroendocrine tumors of lung, adrenal and thyroid, as well as studies on proinsulin to insulin conversion in insulinomas. In addition, practical hints for prevention of background taining, tissue fixation, and silver intensification of gold labeling are given.     

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.     

An enhanced method for post-embedding immunocytochemical staining which preserves cell membranes.

We have devised a method for immunogold staining of unosmicated, plastic-embedded tissue which gives high levels of specific staining without scrificing cell ultrastructure. The key to this method is a combination of several standard techniques optimized to preserve cell membranes as well as antigen. Important conditions include (a) a combination primary fixative, (b) post-fixation with uranyl acetate to preserve membrane phospholipids, (c) dehydration with acetone to minimize extraction of phospholipids, (d) low-temperature embedding in LR Gold resin, and (e) use of osmium tetroxide to stain thin sections after immunogold labeling. We have developed this method specifically to localize the membrane receptor for immunoglobulin G in the jejunal epithelium of the neonatal rat. Ultra-thin sections of embedded tissue were stained with a monoclonal primary antibody and colloidal gold-labeled secondary antibody, followed by 2% osmium tetroxide and lead citrate. The receptor was resolved in the well-preserved network of tubules, endosomes, and other membrane compartments involved in immunoglobulin transport. In several other tissues processed by this method, cell ultrastructure resembled that seen after conventional osmium post-fixation and epoxy embedding. In addition to its usefulness in these studies, this general method should be applicable to many other immunocytochemical problems.     

A Comparison of Methods for Heat-Mediated Antigen Retrieval for Immunoelectron Microscopy: Demonstration of Cytokeratin No. 18 in Normal and Neoplastic Hepatocytes

Postembedding antigen retrieval is a veil established technique for immnoelectron microscopy; however, many antigens cannot be detected without additional unmasking procedures. This study was undertaken to determine whether microwave oven heating, autoclaving, and pressurized boiling, which are well recognized methods of antigen retrieval for light microscopy, and simple boiling can also be used in electron microscopy. We investigated neoplastic and normal hepatocytes using a commercially available mouse monoclonal antibody against cytokeratin NO. 18 (CK18). The tissue was fixed in paraformaldehyde / gintaraldehyde and embedded in Lowicryl K4M at -40 C. Ultrathin sections in various buffers were exposed to heat using one of four methods or to pronase at 37 C before incubation with the primary antibody. The secondary antibody was gold-labeled goat anti-mouse antibody. Sections that were not heat-treated remained unlabeled, but heat-treated sections showed immu-noreactivity located mainly at the cytoplasmic periphery. Some of the gold particles lay in direct or loose association with intermediate filaments, some were seen in the area of desmosomes, and some did not appear related to any structures. No difference in immunostainlng was found among the four methods of heat treatment. The citrate buffer, pH 6.0, and 10 mM EDTA, pH 8.0, generated the best labeling results.     

A Comparison of Methods for Heat-Mediated Antigen Retrieval for Immunoelectron Microscopy: Demonstration of Cytokeratin No. 18 in Normal and Neoplastic Hepatocytes

Postembedding antigen retrieval is a veil established technique for immnoelectron microscopy; however, many antigens cannot be detected without additional unmasking procedures. This study was undertaken to determine whether microwave oven heating, autoclaving, and pressurized boiling, which are well recognized methods of antigen retrieval for light microscopy, and simple boiling can also be used in electron microscopy. We investigated neoplastic and normal hepatocytes using a commercially available mouse monoclonal antibody against cytokeratin NO. 18 (CK18). The tissue was fixed in paraformaldehyde / gintaraldehyde and embedded in Lowicryl K4M at -40 C. Ultrathin sections in various buffers were exposed to heat using one of four methods or to pronase at 37 C before incubation with the primary antibody. The secondary antibody was gold-labeled goat anti-mouse antibody. Sections that were not heat-treated remained unlabeled, but heat-treated sections showed immu-noreactivity located mainly at the cytoplasmic periphery. Some of the gold particles lay in direct or loose association with intermediate filaments, some were seen in the area of desmosomes, and some did not appear related to any structures. No difference in immunostainlng was found among the four methods of heat treatment. The citrate buffer, pH 6.0, and 10 mM EDTA, pH 8.0, generated the best labeling results.     

Combined effects of formalin fixation and tissue processing on immunorecognition

Abstract

It is accepted that aldehyde-based fixation of cells can affect immunodetection of antigens; however, the effects of tissue processing on immunodetection have not been analyzed systematically. We investigated the effects of aldehyde-based fixation and the various cumulative steps of tissue processing on immunohistochemical detection of specific antigens. DU145 (prostate) and SKOV3 (ovarian) cancer cell lines were cultured as monolayers on microscope slides. Immunohistochemical detection of Ki67/MIB-1 and proliferating cell nuclear antigen (PCNA) was evaluated after various fixation times in 10% neutral buffered formalin and after each of the several cumulative steps of tissue processing. The effect of antigen retrieval (AR) was evaluated concomitantly as an additional variable. Our results indicate that in addition to fixation, each of the tissue processing steps has effects on immunorecognition of the epitopes recognized by these antibodies. Extensive dehydration through ethanols to absolute ethanol had only modest effects, except for the detection of Ki67/MIB-1 in SKOV-3 cells where the effect was stronger. In general, however, establishment of a hydrophobic environment by xylene resulted in the greatest decrease in immunorecognition. AR compensated for most, but not all, of the losses in staining following fixation and exposure to xylene; however, AR gave consistent results for most steps of tissue processing, which suggests that AR also should be used for staining PCNA. The cellular variations that were observed indicate that the effects of fixation and other steps of tissue processing may depend on how antigens are packaged by specific cells.     

Phalloidin-eosin followed by photo-oxidation: a novel method for localizing F-actin at the light and electron microscopic levels.

We describe a novel high-resolution method to detect F-actin at the light and electron microscopic levels through the use of the actin-binding protein phalloidin conjugated to the fluorophore eosin, followed by photo-oxidation of diaminobenzidine. This method possesses several key advantages over antibody-based labeling and structural methods. First, phalloidin binding to F-actin can tolerate relatively high concentrations of glutaraldehyde (up to 1%) in the primary fixative, resulting in good ultrastructural preservation. Second, because both eosin and phalloidin are relatively small molecules, considerable penetration of reagents into aldehyde-fixed tissue was obtained without any permeabilization steps, allowing 3D reconstructions at the electron microscopic level. By employing a secondary fixation with tannic acid combined with low pH osmication, conditions known to stabilize actin filaments during preparation for electron microscopy, we were able to visualize individual actin filaments in some structures. Finally, we show that fluorescent phalloidin can be directly injected into neurons to label actin-rich structures such as dendritic spines. These results suggest that the fluorescent phalloidin is an excellent tool for the study of actin networks at high resolution.     

Visualization of identified GFP-expressing cells by light and electron microscopy.

We have developed a procedure for visualizing GFP expression in fixed tissue after embedding in LR White. We find that GFP fluorescence survives fixation in 4% paraformaldehyde/0.1% glutaraldehyde and can be visualized directly by fluorescence microscopy in unstained, 1 microm sections of LR White-embedded material. The antigenicity of the GFP is retained in these preparations, so that GFP localization can be visualized in the electron microscope after immunogold labeling with anti-GFP antibodies. The ultrastructural morphology of tissue fixed and embedded by this protocol is of quality sufficient for subcellular localization of GFP. Thus, expression of GFP constructs can be visualized in living tissue and the same cells relocated in semithin sections. Furthermore, semithin sections can be used to locate GFP-expressing cells for examination by immunoelectron microscopy of the same material after thin sectioning.     

Dynamics and visualization of MCF7 adenocarcinoma cell death by aptamer-C1q-mediated membrane attack

This study was designed to characterize binding of a DNA aptamer to breast cancer cells and to test whether that aptamer could be used to kill target cells in vitro as part of an aptamer-C1q protein conjugate by coupling to the classic complement cascade. A biotinylated DNA aptamer designated MUC1-5TR-1 was shown to decorate the plasma membranes of human breast adenocarcinoma (MCF7) cells via fluorescence confocal microscopy. Biotinylated aptamer binding successfully initiated the classical complement pathway leading to complement fixation on the target cells via a streptavidin-C1q conjugate as previously reported. F?rster Resonance Energy Transfer (FRET) measurements demonstrated membrane depolarization upon aptamer binding, providing indirect evidence of membrane attack complex (MAC) formation as a result of aptamer binding. Transmission electron microscopy (TEM) and immunogold labeling confirmed that aptamer-mediated complement fixation results in MAC formation on the plasma membrane, leading to osmotic swelling and cell death. This approach may provide a much less toxic and more precisely targeted "antibody-like" treatment for cancers by coupling to the patient's innate immune system in much the same way as more expensive humanized monoclonal antibodies.     

Localization of lanthanum tracer in oral epithelium using transmission electron microscopy and the electron microprobe

Although water soluble tracers have been used to localize intercellular permeability barriers with the transmission electron microscope, there is the possibility of translocation or loss of the tracer during processing. This study compares the localization of lanthanum tracer in keratinized oral epithelium after routine processing with lanthanum seen after using freeze drying to avoid aqueous fixation and dehydration. The electron probe was used to identify the lanthanum tracer in the tissue and to distinguish it from other electron-dense material. After incubating small biopsies of rat palate mucosa in 1% lanthanum nitrate, specimens were either routinely processed for electron microscopy or quick frozen, dehydrated, fixed in osmium vapour and infiltrated with epoxy resin. Examination in the transmission electron microscope indicated that preservation of the freeze dried tissue did not compare favourably with that of normally processed tissue, but the distribution of the electron-dense tracer in the intercellular spaces and the extent of the penetration through the epithelia was similar in the two types of preparations. Confirmation of the tracer as lanthanum was obtained by wavelength dispersive X-ray analysis with the electron probe. The results indicate that no appreciable shift in the localization of the tracer is introduced by routine aqueous fixation and dehydration for electron microscopic examination.