pH-dependent effect of heat-induced antigen retrieval of epoxy section for electron microscopy

The aim was to examine how the pH in the antigen retrieval medium (citrate) affects the yield of immunogold labeling of epoxy sections. Renal swine tissue with glomerular immune complex deposits with reactivity against IgG was embedded in epoxy resin. Prior to immunogold labeling with anti-IgG, ultrathin sections from these blocks were exposed to antigen retrieval by heating in citrate solution (pH 6, 9 or 12) at 95 degrees C in a PCR-machine or at 121 or 135 degrees C min in an autoclave. The level of immunogold labeling was significantly higher for pH 12 than for pH 6 when heated at 95 degrees C (50% more intense), but at the cost of the ultrastructural preservation of the tissue. At pH 12 and temperature 135 degrees C the epoxy sections were completely destroyed. The sections which had been heated at 135 degrees C, pH 6 appeared significantly better both with respect to intensity of immunogold labeling (85% more intense) and to ultrastructural preservation than those which were heated at 95 degrees C, pH 12. Therefore, our results indicate that relatively low pH (pH 6) and high temperature is the method of choice, but low temperature and high pH can be used when an autoclave is not available.     

Comparison of the immunolabeling of heated and deplasticized epoxy sections on the electron microscopical level.

The purpose of this study was to compare the yield of immunogold labeling of heated epoxy sections with the yield of labeling of deplasticized epoxy sections, and to compare the immunolabeling of deplasticized high-accelerator epoxy sections and deplasticized low-accelerator epoxy sections. Renal swine tissue and human thyroid tissue were embedded in both high- and low-accelerator epoxy resin and also in LR-White resin. Immunogold labeling was performed on deplasticized (ethoxide-treated), heated and non-treated ultrathin sections from these specimens. The renal tissue was immunolabeled with anti-IgG, and the thyroid tissue was immunolabeled with anti-thyroglobulin. The ethoxide treatment of the epoxy sections induced complete deplasticizing. The immunogold labeling with anti-IgG on deplasticized epoxy sections of renal tissue demonstrated significantly more intense immunolabeling of immune complex deposits than the corresponding epoxy sections which were exposed to heat in citrate buffer. The results for labeling areas of thyroglobulin substance with anti-thyroglobulin showed no significant differences between deplasticized and heated epoxy sections, probably because the sodium ethoxide partly destroys the antigenicity. Deplasticized high-accelerator epoxy sections showed significantly higher yield of immunolabeling than deplasticized low-accelerator epoxy sections and LR-White sections both for anti-IgG and anti-thyroglobulin. This can be explained by the reduced tendency for the knife to cleave proteins when cutting high-accelerator epoxy sections. High-accelerator epoxy sections which were exposed to heat in citrate buffer were more intensely immunolabeled than similarly treated low-accelerator epoxy sections, in agreement with previous results. The ultrastructural preservation of the tissues of deplasticized epoxy sections was inferior compared with the other sections. This study shows that the choice between deplasticizing technique or heating of epoxy sections has to be considered with respect to the nature of the antigen and to the requirement for ultrastructural preservation.     

Increased level of immunogold labeling of epoxy sections by rising the temperature significantly beyond 100 degrees C in the antigen retrieval medium

The purpose of this study was to compare the level of immunogold labeling of epoxy sections when the sections were subjected to antigen retrieval at different temperatures. Renal swine tissue with glomerular immune complex deposits with reactivity against IgG and C3 was embedded in epoxy resin. Sections from these blocks were exposed to antigen retrieval by heating in citrate solution at temperatures in the range of 25-135 degrees C. Immunogold labeling with anti-IgG and anti-C3 was performed on the heated sections. The level of immunogold labeling increased significantly in the direction of increased heat. Interestingly, the level of immunogold labeling was significantly higher when exposed to heating in the autoclave (121 and 135 degrees C) than at temperatures just below the normal boiling point. Sections stained with anti-C3 turned from almost negative labeling when heated at 95 degrees C to strong positive labeling when heated at 135 degrees C (11 times increased). The intensity of the immunogold labeling with anti-IgG increased almost three times when raising the temperature in the retrieval medium from 95 to 135 degrees C. The practical significance of these results is that antigen retrieval of epoxy sections should be performed by heating in aqueous solutions at 135 degrees C or higher to obtain maximum immunolabeling.     

A comparative study of the immunogold labeling on H(2)O(2)-treated and heated epoxy sections.

The purpose of this study was to compare the intensity of the immunogold labeling of H(2)O(2)-treated and heated epoxy sections. Renal swine tissue with glomerular immune complex deposits with reactivity against IgG was embedded in epoxy resin. Immunogold labeling with anti-IgG was performed on sections from these blocks. Some of these sections were treated by H(2)O(2), others were heated in a citrate solution, while some were not treated at all. Some epoxy sections, which had been exposed to both H(2)O(2) and heat, were also exposed to the same immunolabeling. The heated epoxy sections obtained an yield of specific immunogold labeling, which was twice as large as the labeling of the H(2)O(2)-treated sections. The yield of immunolabeling of the sections that had been exposed to both H(2)O(2) and heat was not significantly different from the sections that were only exposed to heat. The non-treated sections were very weakly labeled with anti-IgG. We believe that both H(2)O(2) and heat have the ability to break some chemical bonds between the epoxy resin and the antigens, but heating in citrate buffer has a larger potential in this respect than H(2)O(2). We interpret the results from the combined treatment with H(2)O(2) and heat in the following way; the bonds that are broken by H(2)O(2) will also be broken by heating in citrate solution. The practical significance of these results is that heating in citrate buffer is a more convenient method for enhancing the immunolabeling of epoxy sections than treatment with H(2)O(2).     

Comparison of the immunogold labeling of single light chains and whole immunoglobulins with anti-kappa on LR-white and epoxy sections

The purpose of this study was to examine the intensity of the immunogold labeling of kappa light chains as single molecules and as parts of whole immunoglobulin molecules in LR-White sections and epoxy sections both practically and theoretically. Human renal tissues including deposits of kappa light chains and immune complex deposits of IgA were embedded in both LR-White resin and epoxy resin. Immunogold labeling was performed on unetched thin sections of both resins with anti-κ or anti-IgA. In all relevant cases the immunolabeling was intense on the LR-White sections. Single kappa light chains were intensely labeled also on the epoxy sections, although not as intensely as on LR-White sections. In contrast, the immunogold labeling of whole immunoglobulins with anti-κ and anti-IgA was weak and hardly positive on the epoxy sections. Consequently, the quotient labeling_lr-white/labeling_epoxy for anti-κ was significantly lower for labeling of single light chains (3.6) than for labeling of whole immunoglobulins (15.9). The corresponding quotient for labeling of whole immunoglobulins with anti-IgA was 17.0. Supported by theoretical considerations, it is believed that the copolymerization between the epoxy resin and the antigens allows the knife to cleave the large whole immunoglobulins more easily than the smaller single kappa chains. This prevents satisfactory immunolabeling of whole immunoglobulins on epoxy sections whether anti-κ or anti-IgA is used as antibodies, while single kappa chains are easily immunolabeled.     

The intensity of immunogold labeling of deplasticized acrylic sections compared to deplasticized epoxy sections-Theoretical deductions and experimental data

The purpose of this study was to compare the level of immunogold labeling of deplasticized acrylic sections and deplasticized epoxy sections. Pure protein gels of IgG, albumin and thyroglobulin were produced by glutaraldehyde fixation and embedded in non-crosslinked acrylic resin (Technovit 9100) and epoxy resin (Epon 812), respectively. Ultrathin sections of acrylic and epoxy resin were separately deplasticized in 2-methoxyethyl acetate (MEA) and sodium ethoxide. Quantitative immunogold labeling was performed with anti-IgG, anti-albumin and anti-thyroglobulin antibodies on sections of the corresponding protein gels. For all antibodies tested, the intensity of labeling for deplasticized acrylic sections was significantly higher (two to four times) than for the corresponding deplasticized epoxy sections. The results fit with a theoretically deduced relation: the quotient of the labeling of two deplasticized sections of different resins is equivalent to the square root of the quotient of the labeling of the similar sections not exposed to any kind of pre-treatment. The practical significance of the results is that immunolabeling of deplasticized non-crosslinked acrylic resin results in more intense immunogold labeling than deplasticized epoxy sections. Deplasticizing is most useful when the requirements for ultrastructural preservation according to conventional criteria are moderate. Our theoretically deduced results also indicate that deplasticized Technovit (or other non-crosslinked acrylic resins) sections will be significantly better suited for immunolabeling at the light microscopic level than deplasticized epoxy sections.     

Deplasticizing or etching of epoxy sections with different concentrations of sodium ethoxide to enhance the immunogold labeling.

The study's purpose was to obtain improved "deplasticizing" of epoxy sections for immunoelectron microscopy. Epoxy-embedded renal swine tissue with immune complex deposits was used. Ultrathin sections were mounted on uncoated grids or on carbon-stabilized formvar grids. The sections were exposed to different concentrations of sodium ethoxide, and they were subjected to immunogold labeling with anti-IgG. Etching with > or =8% of saturated solution gave completely deplasticized sections. Sections etched with 2-4% solution were only partly deplasticized, but these sections were detached if mounted on uncoated grids, and the yields of immunolabeling were significantly decreased compared with the deplasticized ones. Sections exposed to < or =1% solution were not detached from the uncoated grids. Double-sided labeling of uncoated sections etched with 1% solution yielded approximately the same immunolabeling as for the completely deplasticized formvar-supported sections, and they gave better ultrastructural preservation of the tissue. We have established that etching epoxy sections on non-supported grids with a diluted solution of sodium ethoxide may be preferable for immunoelectron microscopy.     

A method for measurements of the efficiency of immunogold labelling of epoxy-embedded proteins subjected to different retrieval techniques

The purpose of this study was to compare the level of immunogold labelling of both osmicated and non-osmicated epoxy sections when subjected to different antigen retrieval, etching and incubation temperature for the antibodies. Pure IgG protein gels were produced by glutaraldehyde fixation, eventually postfixed with 1% osmium tetroxide, and embedded in epoxy resin. Ultrathin sections were antigen retrieved in citrate solution at 95 or 144 degrees C and eventually etched with NaIO4. Immunogold labelling with anti-IgG was performed at 4 degrees C overnight or at 60 degrees C for 1 h. The level of labelling for osmicated gels was 140% higher when heated at 144 degrees C and incubated with primary antibodies at 60 degrees C than when heated at 95 degrees C, etched with NaIO4 and incubated with primary antibodies at 4 degrees C. Osmium-fixed IgG-gels antigen retrieved at 144 degrees C and incubated with anti-IgG at 60 degrees C showed more labelling than sections of non-osmicated gels heated at 95 degrees C. Non-osmicated gels gained significant intensity of immunolabelling when the antibody incubation occurred at 60 degrees C for 1 h than at 4 degrees C overnight. Resin embedding of pure protein gels was a useful tool for comparing different protocols for immunoelectron microscopy.     

Morphology and Fracture Toughness of Nanostructured Epoxy Resin Containing Amino-Terminated Poly(propylene oxide)

Amino-terminated poly(propylene oxide) (ATPPO) was incorporated into epoxy resin to toughen thermosets. It was found that nanostructured thermosets were obtained; the nanostructures were characterized by means of atomic force microscopy and small-angle X-ray scattering. The formation of the nanostructures is interpreted on the basis of the occurrence of the reaction of terminal groups of ATPPO with diglycidyl ether of bisphenol A; this reaction is suggested to result in the formation of star-shaped block copolymers composed of poly(propylene oxide) (PPO) and epoxy blocks. Due to the presence of the star-shaped block copolymer produced in situ, the phase separation of PPO induced by the reaction was confined to the nanometer scale. The glass-transition behavior and fracture toughness of the nanostructured thermosets were investigated by means of differential scanning calorimetry, dynamic mechanical thermal analysis, and the measurement of critical stress intensity factors. The epoxy thermosets were significantly toughened by the inclusion of a small amount of ATPPO. The thermal and mechanical properties of the nanostructured thermosets are compared to the binary blends of epoxy resin containing hydroxyl-terminated poly(propylene oxide) (HTPPO) with identical molecular weight. With the identical composition, the nanostructured thermosets displayed higher fracture toughness than that of their binary blends. The difference in morphology and properties is interpreted in terms of the formation of the nanostructures.     

Experimental study of the voids in the electroless copper deposits and the direct measurement of the void fraction based on the scanning electron microscopy images

Electroless copper deposits were plated on epoxy substrates in various plating solutions at either a high operating temperature (60 °C) or a low one (45 °C). Cross section samples were made using epoxy resin cured in room temperature, and then ground, polished and over-etched. The scanning electron microscopy (SEM) images of the over-etched cross section samples show voids in low temperature deposits and solid structure in high temperature ones. The surface morphology images also indicated such structures in low temperature samples. The SEM image of the cross section of a stand-alone deposit prepared on stainless steel substrate shows similar voids observed on etched cross section samples on epoxy board substrates. An image processing program was written using MATLAB to identify the voids in the over-etched cross sections of the deposits from low temperature solutions and thus the void fraction can be directly measured and compared with the previously published simulation results.     

Heat-induced retrieval of immunogold labeling for nucleobindin and osteoadherin from Lowicryl sections of bone

The main purpose of this study was to examine whether antigens can be retrieved by heating Lowicryl sections of paraformaldehyde-fixed (PFF) tissues. Thus the intensity of the immunogold signal for two bone proteins (Nucleobindin (Nuc) and osteoadherin (OSAD)) was compared in retrieved and non-retrieved sections of PFF rat bone. As an additional experiment, the effect of antigen retrieval (for Nuc) in sections of tissue primary stabilized by high pressure freezing with subsequent freeze substitution (HPF-FS) was studied. Finally, the tissue distribution patterns of Nuc labeling were compared in non-retrieved HPF-FS sections to that of retrieved and non-retrieved PFF sections. Antigen retrieval in Lowicryl sections of PFF tissues showed significantly enhanced labeling intensity for both proteins in all compartments where they are known to occur. Retrieved PFF Lowicryl sections showed only minor ultrastructural differences compared to non-retrieved ones. Retrieval of HPF-FS sections exhibited no enhancement of labeling but rather a slight reduction, which was significant in the cytoplasm and in cartilage. Furthermore, striking ultrastructural differences were observed in retrieved HPF-FS sections compared to non-retrieved ones with loss of coherence and structure in sections subjected to heating. Comparison of the distribution patterns of Nuc in the sections of PFF and HPF-FS tissues showed discrepancy in most compartments. Antigen retrieval by heating Lowicryl sections of PFF tissues significantly enhances immunogold labeling in all cell compartments where the bone proteins are known to occur. However, the procedure may distort the tissue distribution pattern of bone proteins.     

A new protocol to prepare dry plant specimens for electron microscopy and immunocytochemistry

Samples from dry kidney bean cotyledons were fixed in acrolein vapor and embedded in Lowicryl K4M using a modified dehydration and embedding technique. The preservation of morphology as investigated by transmission electron microscopy was excellent showing desiccated cells and organelles in life-like preservation. Postembedding immunogold labeling for kidney bean purple acid phosphatase (KbPAP) showed association of kbPAP with ribosome-rich areas of the cytoplasm.     

GFP immunogold staining, from light to electron microscopy, in mammalian cells

GFP has emerged as an important reporter for monitoring gene expression, protein localization, cell transformation and cell lineage. The development of GFP as a marker in many different biological systems has emphasized the need to image GFP at high resolution. GFP immunogold labeling with colloidal gold particles becomes essential for electron microscopy (EM) ultrastructural detection. Because of the small size, colloidal gold particles require silver enhancement, a procedure to increase the size of the particle as well as gold toning to stabilize the silver layer. GFP preembedding immunogold staining enables high quality cellular-ultrastructural EM analysis mainly for two reasons, on one hand it allows adequate fixation for EM analysis maintaining GFP antigenicity, on the other hand it also enables the epoxy resins inclusion after immunogold staining. Both of them help to preserve better the ultrastructure. However GFP immunogold staining presents some drawbacks, such as the progressive decrease in immunogold labeling with tissue depth. Special attention must be taken when using GFP-tagged protein, since the fusion could interfere with their localization and function. In this review we provide a detailed protocol of the GFP immunogold staining, their main applications for EM and possible troubles.     

Fabrication of combined 0–3 and 1–3 connectivities PZT/epoxy resin composites

PZT/epoxy resin composites of combined 0–3 and 1–3 connectivities were fabricated, for the first time, using suction, dice and fill techniques. Two types of composites (PZT(m)/epoxy resin and PZT(sp)/epoxy resin) were produced using PZT powders prepared by mixed oxide and spray-drying methods. Physical, mechanical, dielectric and piezoelectric properties of the composites were examined. Generally, overall results between the two composites were found to be very similar (volumetric changes ∼34–37%, d₃₃∼20.2–25.3 pC/N, K_p∼0.54–0.61). Higher density was found in PZT(sp)/epoxy resin, however, due to better packing of particles. Moreover, both PZT/epoxy resin composites exhibited very low acoustic impedance (Z∼4.12–4.84 Mrayls), which is very close to that of human tissue and water. Therefore, these new composites may be suitable for use in medical applications. PACS 81.05.Qk; 81.05.Zx; 77.87.-s     

Studies on the Thermal Properties of Epoxy Resins Modified with Two Kinds of Silanes

Dimethyldiethoxysilane (DMDES) and diphenyldimethoxysilane (DPDMS)-containing epoxy resins were synthesized by dehydration polycondensation. The chemical structures were determined by FT-IR, ¹H NMR, and ¹³C NMR. The cured samples, with 4, 4′-diaminodiphenylmethane (DDM) as curing agent, were investigated by differential scanning calorimetry (DSC), dynamic mechanical analysis (DMA), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), and tensile and impact testing. Results showed that DMDES and DPDMS-modified epoxy resins possess higher glass transition temperatures, better thermal stability, and better fracture toughness than the neat epoxy resin.     

Shelf Life and Controlled Cure of Epoxies by Loaded Zeolite

Epoxy formulations based on the multi-functional amine hardener, dicyandiamide (Dicy), regularly contain a free accelerator for reducing the curing temperature and the time needed to complete the network formation. Unfortunately, all accelerators reduce the shelf life of these adhesives at 25°C. In order to solve this problem, accelerator-loaded zeolites fillers were developed, optimised with respect to host–guest interactions and characterised by Fraunhofer IFAM (Bremen, Germany) with regard to the release and curing behaviour in epoxy adhesive formulations. They are added to an epoxy adhesive (diglycidylether of bisphenol A (EP) and dicyandiamide (Dicy), mass ratio 100:6.7), stored at 25°C in regular air or cured (heated with β = 10 K/min to 170°C subsequent isothermal curing for 45 min). That shelf life and curing behaviour are investigated by FT-IR spectroscopy and modulated DSC. Compared to the EP containing free accelerator, the zeolite-filled EP possesses a threefold increase in shelf life at 25°C due to the immobilization of the accelerator in the pores of the zeolites. While the free accelerator acts steadily during heating, it is shown that the loaded zeolite releases the accelerator at about 76°C. Surprisingly, the released accelerator is not only involved in the chemical formation of the epoxy network but it accelerates the dissolution of Dicy considerably. As the result, network formation at 170°C finishes after not more than 19 min and the starting temperature for curing could be reduced to 140°C.     

Curing behavior and physical properties of an epoxy nanocomposite with amine-functionalized graphene nanoplatelets

Amine-functionalized graphene nanoplatelets (AGNPs) were prepared via an easy simple one-step process, treating graphite powder with 4-aminobenzoic acid in polyphosphoric acid, and then the effects of the AGNPs on the curing and physical properties of an epoxy resin were studied. The formation of the AGNPs was confirmed by scanning electronic microscopy (SEM), Fourier transform infrared spectroscopy, and thermogravimetric analyzer. Curing behavior of the epoxy/AGNPs nanocomposite was investigated by differential scanning calorimeter. The AGNPs made the epoxide curing reaction with amine groups slightly faster. The physical properties of the epoxy/AGNPs nanocomposite were investigated by dynamic mechanical analyzer, thermomechanical analyzer, and impact test. The AGNPs improved T_g by 21.4 °C, and storage modulus and impact strength of the epoxy resin 23 and 73%, respectively, much more effective than the graphite powder at the same filler loading of 1 phr. SEM images for the fracture surfaces of the epoxy/AGNPs nanocomposite showed improved interfacial bonding between the epoxy matrix and the nanofillers due to the amine functional groups of the AGNPs.     

Nanostructured Thermosetting Blends of Phenolic Thermosets and Poly(ethylene oxide)‐block‐poly(propylene oxide)‐block‐poly(ethylene oxide) Triblock Copolymer

The amphiphilic triblock copolymer, poly(ethylene oxide)‐block‐poly(propylene oxide)‐block‐poly(ethylene oxide) (PEO‐b‐PPO‐b‐PEO) was incorporated into novolac resin to prepare thermosetting blends. The morphology of the thermosetting blends was investigated by means of atomic force microscopy (AFM) and small‐angle x‐ray scattering (SAXS) and the nanostructures were obtained. It was identified that the reaction‐induced phase separation occurred in the blends of phenolic thermosets with the model poly(propylene oxide) (PPO), whereas poly(ethylene oxide) (PEO) was miscible with novolac resin after and before the curing reaction. In terms of miscibility and phase behavior of the subchains of the triblock copolymer with novolac resin, it was demonstrated that the formation of nanostructures in the thermosets followed a mechanism of reaction‐induced microphase separation.     

Compositional contrast of uncoated fungal spores and stained section-face by low-loss backscattered electron imaging

Comparative surface imaging was performed on uncoated fungal spores and stained section-face by field emission scanning electron microscopy with an in-column energy-selective backscattered electron detector. Epoxy resin thin sections (ca. 200 and 500 nm thick) of the osmicated and uranyl acetate/lead citrate-stained fungus were examined with the microscope. Topographical contrast was evident in secondary electron imaging by either a below-lens or an in-lens detector. Meanwhile, low-loss backscattered electron images showed mainly compositional contrast at low accelerating voltages (mostly below 1 kV). With attenuated topographical contrast, several different electron densities could be detected, exhibiting several levels of electron density even on a flat plane of spines. Minute differences in topography on epoxy resin sections as seen by secondary electron imaging represented the periphery of the fungal spores and hyphae. On the other hand, the compositional contrast could be retrieved from stained section-face in low-loss BSE imaging, revealing subcellular entities after contrast inversion. The resolution of low-loss BSE imaging was sufficient to resolve plasma membrane, and various types of vacuoles and vesicles. These results suggest that low-loss backscattered electron imaging could potentially provide compositional information to resolve surface chemical features of uncoated microbial cells and stained section-face with heterogeneous surface compositions.     

Effect of interfacial bonding on impact properties of chopped glass fiber polymer nanocomposites

This paper aims to presents the investigations made on the effect of impact response of chopped glass fiber–epoxy nanocomposite laminates subjected to low velocity impact using instrumented falling weight impact tests. The laminates were prepared using six layers of chopped strand mat with density of 610 gsm with epoxy resin and nanoclay content varied from 1, 3, and 5 wt%, by hand lay-up method. The nanoclay was dispersed into the epoxy by high shear mixing process in order to obtained uniform distribution of individual nanoclay. Laminates were impacted at constant mass with different impact energies. During these low velocity impact tests, the maximum load, absorbed energy, and deflection at peak load were recorded. It was observed that by dispersion of nanoclay as reinforcement, there was significant improvement in load carrying capacity and energy absorption. When a small amount of nanoclay (1 wt%) was added into the epoxy, the maximum load was enhanced by 20%. The presence of stiffer nanoclay significantly reduced the surface cracks propagation and controlled delamination area. Scanning electron microscopy was performed to characterize the damage progression.