Epoxy Sections Research Articles

Immunoelectron microscopy on epoxy sections without deplasticizing to detect glomerular immunoglobulin and complement deposits in renal diseases.

Twenty renal biopsies were studied by immunoelectron microscopy (IEM) after embedding in epoxy resin. Immunogold labeling for immunoglobulins and complement C3 was performed on the epoxy sections, which were not subjected to any kind of etching or deplasticizing prior to the immunolabeling. The concentration of accelerator, DMP-30 (Tri (Dimethyl Amino Methyl) Phenol), was increased in the infiltration and embedding steps far beyond the values normally used to make immunolabeling of these antigens possible on epoxy sections. The sections were stained with tannic acid accompanied by uranyl acetate and lead citrate. Immunofluorescence (IF) for light microscopy was carried out on frozen sections of parallel tissue samples. Some cases with IgA-nephritis demonstrated a higher sensitivity for IEM than IF, in the sense that smaller amounts of antigen were detectable with IEM. Ultrastructural preservation with this method was approximately the same as that usually seen on epoxy-embedded material. By combining excellent immunolabeling with nearly optimal ultrastructural morphology in one procedure, this method is useful particularly in situations where the material available is limited, such as in studies of renal biopsies. As far as we know, this is the first time that immunoglobulins have been satisfactorily immunolabeled on epoxy sections without etching or deplasticizing.

How to Examine the Antigen-damaging Effect of Sodium Ethoxide on Deplasticized Epoxy Sections

The purpose of this investigation was to develop a method that could be used to estimate how damaging sodium ethoxide is to different antigens with respect to immunolabeling when epoxy sections are deplasticized. If we obtain weak labeling for an antigen on deplasticized epoxy sections, this might be caused by the damaging effect of the ethoxide solution. It is therefore interesting to develop a method to check if this really is the reason. Fibrin clots and tissues of human kidney and thyroid were embedded in LR White resin. Some thin sections from these specimen blocks were exposed to sodium ethoxide in the same way as epoxy sections are when being deplasticized. Other sections from the same blocks were not exposed to sodium ethoxide. Both categories of sections were immunogold-labeled with anti-fibrinogen, anti-thyroglobulin, anti-IgA, anti-IgG, or anti-IgM. The intensity of immunolabeling of sections treated with ethoxide was compared with the immunolabeling of corresponding sections that were not treated with ethoxide. No significant differences were found in immunolabeling for fibrinogen, IgA, IgG, and IgM. For thyroglobulin, the intensity was approximately 30% less in tissues that were exposed to sodium ethoxide. The practical significance of this method is that we easily can examine the degree to which a given antigen is affected by sodium ethoxide, which is the agent used for deplasticizing epoxy sections.

The Use of Dimethylsulfoxide for Fixation of Yeasts for Electron Microscopy

Conventional methods of chemical fixation are often inadequate for preserving yeast ultrastructure. The thick cell wall severely limits penetration of fixatives rendering poor detail of the cell wall, membranes, and overall anatomy. Dimethylsulfoxide (DMSO) enhances penetration of chemicals and has been added to fixatives to improve cell preservation. At high concentrations (5 to 50%), however, it affects ultrastructure unpredictably. We found that adding 0.1% DMSO to fixatives greatly improved retention of yeast ultrastructure. Candida albicans, C. glabrata and Aspergillusfumigatus were fixed for 3 hr in 3% paraformaldehyde, 1% glutaraldehyde, 1 mil MgCl2, 1 mM CaCl2, 0.1% DMSO in 0.1 M sodium cacodylate buffer followed by 1% OsO4, 1% K2Cr2O7, 0.85% NaCl. 0.1% DMSO in the same buffer. Thin epoxy sections were post-stained in uranyl acetate and lead citrate. The multilayered character of the cell wall was distinct and well structured. Addition of ruthenium red or alcian blue to the fixatives further enhanced the outer fibrillar layer. The plasma membrane was contiguous and tightly adjacent to the inner manno-protein layer of the cell wall. The cytoplasm was well preserved and the overall preservation of the yeast ultrastructure was significantly improved.

Pyrogallol red-vanadium complex-a new stain for electron microscopy.

We report on the application of a pyrogallol red-vanadium complex (PR-V) for ultracytochemical staining of proteinaceous structures in animal tissues and cell cultures. This dye may be used as a general purpose stain in electron microscopy. In contrast to osmium tetroxide, the price of the material is low and no toxic vapors are produced. The PR-V complex was prepared by addition of vanadium (IV) oxide sulfate to pyrogallol red dissolved in acetate buffer (pH 5.6). The formation of the complex was indicated by a color change from purple-red (lambda max = 520 nm) to violet (lambda max = 539 nm) which occurred at equimolar concentrations of the dye and the metal salt. Under these conditions PR-V was stable for several days. The mechanism of PR-V binding was checked in dot blots using different proteins as well as heparin for control. While heparin remained unstained, proteins were stained in a dose-dependent manner. Deamination of proteins with nitric oxide strongly reduced PR-V staining in dot blots as well as in cell cultures. Optimal staining results of animal cells and tissues were obtained in specimens that had been mildly fixed for at least 1 h or longer with a mixture of 0.1% glutaraldehyde and 1.0% paraformaldehyde dissolved in phosphate-buffered saline, pH 7.2, washed with acetate buffer, pH 5.6, and subsequently treated with PR-V in the presence of 50% ethanol at room temperature. Control specimens without PR-V but treated en bloc with uranyl acetate or sodium molybdate showed similar contrast but less details in the ultrastructure of the tissue. All specimens were embedded in epoxy resin and ultratain sections were stained conventionally with uranyl and lead salt solutions. In electron micrographs, membrane-associated particles, stress fibers and filaments of the cell cortex, collagen fibrils, tight junctions and desmosomes, and other proteinaceous components were clearly visualized only in the PR-V-treated specimens. In conclusion, the ability to bind selectively and specifically to protein-aceous structures makes PR-V a versatile stain to study the localization and distribution of these structures in cells and tissues at the ultrastructural level.

The Insect Flight Muscle Sarcomere as a Model System for Immunolocalization

Highly ordered insect flight muscle provides an excellent system for coordinated immunolocalization of sarcomeric proteins at increasing levels of resolution, from fluorescent and gold-tagged secondary antibodies to 3- and 5-nm gold directly coupled to Fab fragments. The penetration of antibody probes of various sizes into native and preserved muscle or tissue sections is compared. Factors affecting resolution and labeling efficiency are examined, such as probe size, and removal of uncoupled Fc and gold particles. The quality of preservation and the level of structural detail achieved with ice and plastic embedding media and different labeling methods are explored. We discuss problems encountered at the highest level of immunoelectron microscopy using gold/Fab to visualize the probe in relation to well-contrasted,in situmyosin and troponin molecules in 25-nm-thin epoxy sections by transmission EM.

Improved immunogold labeling of epoxy sections by the use of propylene oxide as additional agent in dehydration, infiltration and embedding

The purpose of this study was to examine how the intensity of the immunogold labeling on epoxy sections was affected by the use of propylene oxide as an agent in addition to ethanol in the dehydration and infiltration, and also to examine the effect on the immunogold labeling by adding small amounts of propylene oxide to the embedding mixture. Increased knowledge of the mechanism for antigen detection on resin section was another aim. Thyroid tissue, kidney tissue, and fibrin were embedded in epoxy resin; some with ethanol as the only dehydration agent and others with propylene oxide as an additional agent in dehydration, infiltration or embedding steps in different ways. Immunogold labeling was performed with anti-thyroglobulin, anti-IgG, and anti-fibrinogen, respectively. A higher degree of immunogold labeling was achieved by increasing the concentration of accelerator during infiltration and embedding (Brorson and Skjørten, 1996a, Micron, 27, 211–217). The immunogold labeling of the sections that were based on additional dehydration and infiltration with propylene oxide showed significantly more intense labeling than the section of tissues that had only been exposed to ethanol in the dehydration and infiltration steps. The embedding of tissues in a mixture of epoxy resin and 5–10% propylene oxide gave higher yields of immunogold labeling than if pure epoxy resin was used for the embedding. The improved labeling is explained by higher amplitudes of protruding antigens on the surface of the sections because antigens are less tightly incorporated in the polymer network when using propylene oxide as additional agent in dehydration, infiltration or embedding. These results illustrate the advantage of using propylene oxide as an additional agent when preparing specimens for immunoelectron microscopy with epoxy resin embedding.

Histopathologic and physiologic effects of chronic implantation of microelectrodes in sacral spinal cord of the cat.

Active microelectrodes were implanted for a period of 2 weeks to 3 months into the sacral spinal cord of 10 male cats in order to test the feasibility and the safety of discrete stimulation of the parasympathetic preganglionic nucleus for future clinical applications of microelectrode technology in micturition control. An array of four 50 microns-diameter iridium microelectrodes was inserted beneath the dura in each cat. At weekly intervals, bladder pressure was measured as hydrostatic pressure on an intraluminal catheter. At the end of the period, histopathology was evaluated with serial transverse epoxy sections. Observations included diffuse and focal axonal degeneration in white matter and possible neuronal loss around the electrode in the gray matter, meningeal ensheathment of the shafts, and occasional aseptic inflammation of tissue and apparent movement of the electrodes after implantation. Increased bladder pressure responses to individually pulsed electrodes located within the sacral parasympathetic nucleus were not consistent, and, surprisingly, at least 2 different sites were also effective. As long as 3 months after implantation, in 2 out of 5 animals, pulsing of electrodes consistently produced micturition. We conclude that while microelectrode implants are feasible, further modifications in electrode design are needed to eliminate movement and inflammation.

Improved technique for immunoelectron microscopy. How to prepare epoxy resin to obtain approximately the same immunogold labeling for epoxy sections as for acrylic sections without any etching

The purpose of this study was to improve the immunogold labeling of epoxy sections and to increase our knowledge of the mechanism for how antigens become immunolabeled on resin sections. Tissues from pancreas, thyroid and fibrin clots were embedded in an epoxy resin and LR-White. The epoxy mixture was composed and treated in different ways, especially with respect to altered amounts of accelerator (DMP-30). Immunogold labeling was performed with anti-glucagon, anti-thyroglobulin and anti-fibrinogen respectively. By increasing the amount of DMP-30 in the infiltration steps and/or the embedding step, we observed a significant rise in the immunogold labeling. For the largest proteins the labeling was up to 8 times more intense than the labeling achieved with epoxy sections produced by ‘normal’ amount of accelerator in the embedding mixture and without accelerator in the infiltration mixture. For the smallest protein, glucagon, the differences were almost absent. The labeling of thyroglobulin and fibrinogen on the high accelerator epoxy sections was up to 70% of the labeling of LR-White sections, while conventional epoxy sections showed a labeling of 5–10% of that obtained with acrylic labeling. The cutting qualities of the high-accelerator blocks were similar to that of conventional epoxy embedding. The ultrastructure of the sections from the high-accelerator epoxy blocks were good, and the contrast was improved when tannic acid was used as enhancer. Our theory to explain the improved labeling is that the antigens are less tightly incorporated in the polymer network when the concentration of the accelerator is increased. The method outlined significantly improves the detectability of antigens on epoxy sections, which is the embedding resin routinely used in many laboratories.

The theoretical relationship of immunogold labeling on acrylic sections and epoxy sections

The purpose of this study was to predict the ratio of immunogold labeling of LR-White sections and epoxy sections using theoretical methods. Tissues used in the experiments were pancreas, pituitary, kidney, thyroid and fibrin. Antigens used as test proteins were glucagon, somatostatin, thyroglobulin, chromogranin A, ACTH (adrenocorticotropt hormone), amyloid A and fibrinogen. These are proteins of different sizes. The quotient labeling LR-White/labeling epoxy was deduced theoretically and compared to calculations based on practical immunogold experiments. The theoretically deduced formula showed acceptable correlation to these calculations. This study gives a theory—expressed mathematically—for what is happening on the molecular level at the surface of resin sections in immunoelectron microscopy. The theory explains why acrylic resins normally are better suited for immunoelectron microscopy than epoxy sections, and indicates increased usefulness of epoxy sections when the diameter of the protein carrying the epitope decreases.

The theoretical ratio of immunogold labeling of deplasticized epoxy sections and acrylic sections

The purpose of this study was to predict the ratio of immunogold labeling of deplasticized epoxy sections and LR-White sections on the basis of theoretical considerations. Tissues used in the experiments were pancreas, pituitary, kidney, thyroid, and fibrin. Antigens used as test proteins were glucagon, somatostatin, thyroglobulin, chromogranin A, ACTH (=Adrenocroticotropt hormone), amyloid A, and fibrinogen. These are proteins of different sizes. The quotient labeling deplasticized/labeling LR-white was deduced theoretically and compared to measurements based on immunogold experiments to obtain a theoretical model with acceptable correlation to the measurements. This study describes a theory—expressed mathematically—for what happens at the molecular level in immunoelectron microscopy at the surface of deplasticized epoxy sections and acrylic sections. The theory explains why we normally get about the same amount of immunogold labeling using LR-White sections (acrylic resin) and deplasticized epoxy sections. Taking the nuances into account, the theory indicates increased usefulness of deplasticized epoxy sections when the diameter of the protein carrying the epitopes decreases.

Tissue compartments in laminae II-V of rabbit visual cortex--three-dimensional arrangement, size and developmental changes.

The neuropil in laminae II/III and IV of the mature rabbit visual cortex is subdivided into (a) dendrite bundles consisting of apical dendrites of pyramidal cells and associated axons and glial processes, (b) bundles of myelinated axons ascending vertically from the white matter up to lamina IV and (c) neuropil between bundles comprising mainly thin unmyelinated axons, small dendrites and associated glial processes. In this investigation the three-dimensional structure of these compartments was analysed. In addition, the volume fractions of the three neuropil compartments, perikarya and blood vessels in the different laminae and their quantitative changes from the late fetal period up to young adulthood and in a group of aged animals were determined. Serial 1-micron epoxy sections were analysed. Dendrite bundles are more numerous and more intensively intertwined in lamina II/III than in lamina IV. At 28 days after conception the tissue in laminae II-V consists of approximately equal amounts, i.e. between 40 and 50%, of perikarya and neuropil. The volume fraction of blood vessels is about 4% and does not change much during development. During the first 16 days after birth the volume fraction of the neuropil increases to more than 70%, and conversely the volume fraction of nerve cells decreases to about 20%. Later, significant changes are seen only when the volume fractions of the three neuropil compartments are considered separately. The volume fraction of the neuropil between bundles increases throughout all laminae investigated, whereas the volume fraction of dendrite bundles is found to decrease. After 1 month, in lamina IV further increase of the neuropil between bundles is less marked, because here the bundles of myelinated axons become visible as an additional compartment. In young adult animals, the volume fractions of dendrite bundles are about 28% in the upper half, 16% in the lower half of lamina II/III and 7% in lamina IV. The neuropil between bundles comprises about 52% in the upper half, 65% in the lower half of lamina II/III and 62% in lamina IV. In lamina IV 14% is occupied by bundles of myelinated axons. In aged animals, the volume fraction of the neuropil between bundles decreases significantly in all laminae investigated. From previous ultrastructural studies, the extracellular space is known to be about 30% larger in the neuropil between bundles than in the dendrite bundles. Moreover, due to the prevalence of thin cell processes, the degree of tortuosity is larger in the neuropil between bundles than in the dendrite bundles. The present results together with these previous data are an indication of lamina-specific differences in the structure of the extracellular space. This may help to explain the electrical conductivity in the respective laminae of the cerebral cortex. The time course of postnatal changes of the neuropil compartments coincides with fundamental steps of structural and functional maturation of the rabbit visual cortex that are documented in the literature, and thus may be a valid parameter to investigate the degree of maturation or aging by morphological means.

Use of Floor Polish in Mounting Sections for Light Microscopy

Abstract Over the years a number of methods have been described to mount tissue sections on glass slides (for example see Humason 1979). This paper presents a novel mounting medium that is inexpensive and readily available at the nearest grocery store - floor polish. This floor polish is available under a variety of trade names (e.g., Brite® and Future® both from S.C. Johnson and Son. Racine, WI) labeled for use with "no-wax" floors. It has been found that this product is well suited to use as a mounting medium for both gelatin/albumin embedded sections as well as epoxy sections. The floor polish has demonstrated a much lesser degree of tissue distortion in some tissues as compared to the mounting media previously used in our laboratory, including Permount® (Fisher Chemical, Fair Lawn, NJ) and 30% sucrose in distilled H2O.

Mechanism for antigen detection on deplasticized epoxy sections

The purpose of this investigation was to explain why deplasticizing of epoxy sections gives higher immunogold labeling than non-deplasticizing. The methods used were the following: (1) Comparison of the ratio of immunogold labeling of deplasticized and non-deplasticized sections with gold particles of different sizes and comparison of this ratio with respect to sections of different thickness, (2) the tilt method (Brorson et al., 1994).Human kidney tissue with amyloid A depositions, human fibrin, and human pituitary tissue were embedded, sections were deplasticized on grids, treated with anti-Aa, anti-fibrinogen or anti-ACTH (ACTH=adrenocorticotropic hormone), and reembedded on grids. Indications of significant antibody penetration were found only at the periphery of structures (ACTH-vesicles). This penetration was about 30 nm. The ratios of immunogold labeling of deplasticized and non-deplasticized sections were approximately 2, 5 and 1 for amyloid, fibrin and ACTH, respectively, and were independent of the gold particle size. No significant differences of gold labeling were found between thicker and thinner deplasticized epoxy sections regardless the gold particle size. No significant differences of gold labeling between deplasticized epoxy sections and LR-White sections were found on interior areas of ACTH-vesicles or amyloid A plaques. The increased labeling of deplasticized epoxy sections compared to normal epoxy sections seemed to be mainly a surface phenomenon. The practical significance of this observation is that deplasticizing of epoxy sections may be a better method for localizing antigens at the periphery of structures than the use of other resin embedding media. Deplasticizing of epoxy sections may be a method of choice in a pathological laboratory to detect antigens in routinely embedded tissues.

On the real structure of the cytoplasmic matrix: learning from the embedment-free electron microscopy.

The recent debate on the nature of the cytoplasmic matrix is reviewed on the basis of results obtained by electron microscopy of embedment-free materials, i.e., the critical point-dried whole mount-cell method, the polyethylene glycol (PEG)-embedding and subsequent de-embedding section method, and the freeze-etching method. Fine structural images obtained by these methods are carefully evaluated and close correspondence with electron microscopy regardless of these methods is demonstrated. Especially, 'novel' filamentous structures--the microtrabecular strands and the cross-linkers--correspond well to each other; they are structures which have been included in epoxy sections by conventional methods, but have been obscured simply because of a similar property of electron scattering between the filamentous structures and epoxy resin. Although this correspondence seems to support the existence of the microtrabeculae, the electron microscopy of serum albumin, when processed by the PEG-method, also exhibits filamentous networks resembling the microtrabecular lattice. This, together with the finding on the centrifugation of in situ cells, strongly suggests a possibility that most, if not all, microtrabecular strands and cross-linkers in cells without pretreatment by detergents do not represent actual in situ structures.

Radioautographic Study on the Intracellular Localization of Bezafibrate in Cultured Rat Hepatocytes.

In order to demonstrate the intracellular localization of the peroxisome proliferator in the hepatocytes, we have examined the localization of silver grains due to 14C-labeled bezafibrate in cultured rat hepatocytes by means of light and electron microscopic radioautography. On light microscopic radioautograms of the epoxy resin sections at each incorporation period, about 90% of all the silver grains were localized over the cytoplasmi Then, statistical significance was evaluated on grain density in the cytoplasm. On electron microscopic radioautograms of whole mount cultured cells observed by high voltage electron microscopy, silver grains were localized on the cytoplasmic matrix, especially over the endoplasmic reticulum. From these results, it is concluded that bezafibrate was localized over the endoplasmic reticulum. This fact suggests than the receptor of peroxisome proliferator should be associated with the endoplasmic reticulum or the receptor exists on the endoplasmic reticulum. Thus, it is demonstrated that the peroxisome proliferator acts on the endoplasmic reticulum of hepatocytes to proliferate peroxisomes.

Staining etched epoxy resin sections for light microscopy.

Staining of etched sections for light microscopy is described. Azan staining was successful after treatment with potassium dichromate and the use of concentrated dye solutions. To remove osmium for hematoxylin-eosin staining, removal by reduction with ferrocene was used instead of oxidation. Highly selective differentiation after hematoxylin staining was achieved using p-toluenesulfonic acid-DMSO. To enhance eosin staining, a 2-bromoethylamine link between eosin and the tissue was used. Ferrocene also facilitated counterstaining of nuclei with hematoxylin after the PAS reaction. Periodic acid-methenamine silver staining was carried out without modification.

Silver enhancement of colloidal gold particles in deplasticized semithin epoxy sections.

A simple method for light microscope screening of pre-embedded, gold-labeled samples, by silver enhancing deplasticized semithin epoxy sections is presented. This method is of great help in visualizing the topography of gold labeled sites at low magnification and for selecting labeled sample regions for subsequent electron microscope analysis on serial ultrathin sections. The method was developed during a study of lectin binding sites on the egg surface of the anuran amphibian Discoglossus pictus.

The design of a control system for a superimposed frequency fatigue test machine: Stoten, D.P. and Smith, D.J. ASTM J Test Eval (May 1993) 21 (3), 154–159

The experiments reported here were conducted using the framework of the strain-space formulation of plasticity. A servohydraulic multiaxial testing machine was used to perform combined axial-torsional experiments on a thin-walled cylindrical aluminum specimen. The specimen was machined from extruded aluminum 1100 tubing, and annealed at 650 °F for 1 h. Conical epoxy sections were molded on the ends of the specimen for gripping. The specimen was loaded at strain rates under 20×10−6 per minute. An offset strain of 5×10−6 was used to define yield. A novel technique for identifying yield surfaces is introduced, which makes use of an experimentally measured focal point through which all yield probes are made to pass. Yield surfaces were obtained both in stress space and in strain space at various stages of plastic deformation. Although all strains were under 1%, the inelastic states included situations in which the origin in stress space lies outside the elastic region. The yield surfaces in both spaces were found to change shape considerably with deformation, usually developing a region of high curvature near the preload point and a region of low curvature on the opposite side of the yield surface. Experiments were also designed and conducted to test the validity of a prescription for identifying plastic strain in the context of the strain-space formulation. Changes in plastic strain were induced under incremental loading conditions and measured using the prescription. To within experimental error, the measured plastic strain increments satisfy the normality condition. The plastic strain increments are consistent with a flow rule of the type postulated in the strain-space formulation of the theory. The experimental results lend support to the constitutive theory and to the prescription for identifying plastic strain.

Elevated temperature fatigue crack growth in alloy 718. I. Effects of mechanical variables: Ghonem, H., Nicholas, T. and Pineau, A. Fatigue Fract Eng mater Struct (May 1993) 16 (5), 565–576

The experiments reported here were conducted using the framework of the strain-space formulation of plasticity. A servohydraulic multiaxial testing machine was used to perform combined axial-torsional experiments on a thin-walled cylindrical aluminum specimen. The specimen was machined from extruded aluminum 1100 tubing, and annealed at 650 °F for 1 h. Conical epoxy sections were molded on the ends of the specimen for gripping. The specimen was loaded at strain rates under 20×10−6 per minute. An offset strain of 5×10−6 was used to define yield. A novel technique for identifying yield surfaces is introduced, which makes use of an experimentally measured focal point through which all yield probes are made to pass. Yield surfaces were obtained both in stress space and in strain space at various stages of plastic deformation. Although all strains were under 1%, the inelastic states included situations in which the origin in stress space lies outside the elastic region. The yield surfaces in both spaces were found to change shape considerably with deformation, usually developing a region of high curvature near the preload point and a region of low curvature on the opposite side of the yield surface. Experiments were also designed and conducted to test the validity of a prescription for identifying plastic strain in the context of the strain-space formulation. Changes in plastic strain were induced under incremental loading conditions and measured using the prescription. To within experimental error, the measured plastic strain increments satisfy the normality condition. The plastic strain increments are consistent with a flow rule of the type postulated in the strain-space formulation of the theory. The experimental results lend support to the constitutive theory and to the prescription for identifying plastic strain.

Axial-torsional fatigue: a study of tubular specimen thickness effects: Bonacuse, P.J. and Kalluri, S. ASTM J Test Eval (May 1993) 21 (3), 160–167

The experiments reported here were conducted using the framework of the strain-space formulation of plasticity. A servohydraulic multiaxial testing machine was used to perform combined axial-torsional experiments on a thin-walled cylindrical aluminum specimen. The specimen was machined from extruded aluminum 1100 tubing, and annealed at 650 °F for 1 h. Conical epoxy sections were molded on the ends of the specimen for gripping. The specimen was loaded at strain rates under 20×10−6 per minute. An offset strain of 5×10−6 was used to define yield. A novel technique for identifying yield surfaces is introduced, which makes use of an experimentally measured focal point through which all yield probes are made to pass. Yield surfaces were obtained both in stress space and in strain space at various stages of plastic deformation. Although all strains were under 1%, the inelastic states included situations in which the origin in stress space lies outside the elastic region. The yield surfaces in both spaces were found to change shape considerably with deformation, usually developing a region of high curvature near the preload point and a region of low curvature on the opposite side of the yield surface. Experiments were also designed and conducted to test the validity of a prescription for identifying plastic strain in the context of the strain-space formulation. Changes in plastic strain were induced under incremental loading conditions and measured using the prescription. To within experimental error, the measured plastic strain increments satisfy the normality condition. The plastic strain increments are consistent with a flow rule of the type postulated in the strain-space formulation of the theory. The experimental results lend support to the constitutive theory and to the prescription for identifying plastic strain.