Biomedical subjects

S H Brorson

Publications and source records attributed to S H Brorson.

33 records · Page 2Linked to original sources

PubMed1997

Bovine serum albumin (BSA) as a reagent against non-specific immunogold labeling on LR-White and epoxy resin.

The purpose of this study was to examine how different incubation times with different concentrations of bovine serum albumin (BSA) affect the amount of non-specific immunogold labeling on epoxy sections and LR-White sections. Immunogold labeling was performed on epoxy sections and LR-White sections of renal tissue with IgG-deposits and fibrin clots, and the antibodies used were anti-IgG and anti-fibrinogen, respectively. The sections were incubated with different concentrations of BSA prior to application of primary antibodies, and the length of this pre-incubation step varied between 0 and 4 h. During the incubation with primary antibodies, BSA was added in the same concentration as in the pre-incubation step. The results showed that the non-specific labeling on the resin decreased significantly when the concentration of BSA or the length of the preincubation step was increased. The non-specific labeling was usually higher on the epoxy resin than on the LR-White resin when using the same conditions with respect to BSA. But, when the preincubation step with BSA lasted 4 h, the non-specific labeling was somewhat lower on epoxy resin than on the acrylic LR-White resin, without respect to the concentration of BSA. The specific labeling for both fibrinogen and IgG decreased slightly when the concentration of BSA and incubation time increased, probably due to the steric hindrance performed by BSA molecules on the section. Blocking procedures with at least 1 h incubation time for the blocking step with at least 5% BSA are recommended for both epoxy and LR-White sections.

Acrylic Resins↗

PubMed1997

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.

Complement C3c↗

PubMed1997

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.

Acrylic Resins↗

PubMed1996

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 sections 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 sections 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.

Animals↗

PubMed1995

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.

Adrenocorticotropic Hormone↗

PubMed1994

Antibody penetration into LR-White sections.

The purpose of this investigation is to study the ability of antibodies to penetrate sections of LR-WHITE resin. The methods used in this study were the following: (1) Reembedding of sections labeled with immunogold (1 nm) and peroxidase/DAB/gold chloride, (2) tilting of ultrathin sections treated with immunogold (1 nm), (3) immunolabeling of cylindrical structures embedded in LR-WHITE, (4) application of primary and secondary antibodies on opposite sides of ultrathin sections. Fibrin and human pituitary tissue was embedded in LR-WHITE and treated with anti-fibrinogen or anti-ACTH respectively (ACTH = Adrenocorticotropic hormone). No indication of antibody penetration into the section were found with either of the methods, contrary to findings in earlier publications. The significance of this result is that antigens cannot be demonstrated in the interior of LR-WHITE sections with post-embedding techniques. Furthermore, LR-WHITE resin may be used for quantitative immunoelectron microscopy, and the resin may be used for double immunogold labeling since the application of immunoreagents on opposite sides of the sections is completely safe.

Acrylic Resins↗

PubMed1992

The use of post-embedding immunoelectron microscopy in the diagnosis of glomerular diseases. Comparison of immunoelectron microscopic and immunofluorescence studies.

Fifty renal biopsies were studied by immunoelectron microscopy after embedding in a partly hydrophilic polyacrylic resin (LR White). Immunofluorescence studies were carried out on frozen sections of parallel tissue samples. Polyacrylic embedding gave good preservation of the renal ultrastructure and precise localization of immunoglobulin and C3c antibodies within glomerular electron-dense deposits. Non-specific staining of plasma proteins within vascular lumina could easily be detected. There was good correlation between immunoelectron and immunofluorescence microscopy. Immunoelectron microscopy is a very sensitive method, which can detect small amounts of antigen. More cases were, however, positive by immunofluorescence than by immunoelectron microscopy. This discrepancy may be explained by difference in sample size, and by difference in resolution of morphological details (electron microscopy versus fluorescence microscopy).

Acrylic Resins↗

PubMed1992

Neuronal uptake of plasma proteins in cryogenic brain lesions. An immunoelectron microscopic study.

A previous light microscopic study on cryogenic brain lesions in rats demonstrated uptake of plasma proteins into damaged neurons within a few minutes after the lesion. The protein concentration was much higher inside the nerve cell bodies than in the surrounding neuropil. This is puzzling since the neuropil to a large extent consists of damaged neuronal processes. The present investigation describes the intracellular localization of albumin in this model using a post-embedding immunoelectron microscopic technique. The distribution of albumin in the lesions was studied after 1, 6 and 12 h survival periods. The intraneuronal albumin was mainly bound to the particulate elements of the cytoplasm and nuclei, while the watery parts of the cells showed no immunoreactivity. The intracellular organelles contained very little albumin, indicating that their membranes may be more resistant to freezing than those of the cells. Most of the neuronal and glial processes in the neuropil were swollen and contained almost no albumin. This explains the contrast between the strong immunoreactivity of the neurons and the vague reactivity of the neuropil in light microscopy.

Animals↗

PubMedDate not supplied

Transformation of cystic forms of Borrelia burgdorferi to normal, mobile spirochetes.

The purpose of this study was to evaluate the behaviour of Borrelia burgdorferi under controlled conditions. The occurrence of cystic forms of Borrelia burgdorferi in vitro was noted, and these cysts were able to be transformed to normal, mobile spirochetes. B. burgdorferi was cultivated in a commercial culture medium without serum. The spirochetes multiplied only slowly in this medium, and transformation to encysted forms was observed after 1 week. When these cysts were transferred to the same culture medium with rabbit serum, the encysted forms developed into regular, mobile spirochetes after 6 weeks, and their regeneration time was normal. Examination of these cysts in the transmission electron microscope revealed transverse fission inside the cysts. It is probable that similar phenomena may occur in vivo under conditions unfavourable for spirochetes. These observations may help to explain why diagnosis and treatment of B. burgdorferi infections in humans can be difficult.

Animals↗

PubMedDate not supplied

In vitro conversion of Borrelia burgdorferi to cystic forms in spinal fluid, and transformation to mobile spirochetes by incubation in BSK-H medium.

The purpose of this study was to examine the structural alterations of Borrelia burgdorferi when exposed to spinal fluid. Normal, mobile spirochetes were inoculated into spinal fluid, and the spirochetes were converted to cysts (spheroplast L-forms) after 1-24 h. When these cystic forms were transferred to a rich BSK-H medium, the cysts were converted back to normal, mobile spirochetes after incubation for 9 to 17 days. The cultures were examined by dark field microscopy (DFM), interference contrast microscopy (ICM) and transmission electron microscopy (TEM). When neuroborreliosis is suspected, it is necessary to realize that B. burgdorferi can be present in a cystic form, and these cysts have to be recognized by microscopy. This study may also explain why cultivation of spinal fluid often is negative with respect to B. burgdorferi.

Borrelia Infections↗

PubMedDate not supplied

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 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 achieve 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.

Acrylic Resins↗

PubMedDate not supplied

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 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 labelingdeplasticized/labelingLR-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.

Acrylic Resins↗

PubMedDate not supplied

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 labelingLR-White/labelingepoxy 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.

Acrylic Resins↗

PubMedDate not supplied

The combination of high-accelerator epoxy resin and antigen retrieval to obtain more intense immunolabeling on epoxy sections than on LR-white sections for large proteins.

The purpose of this study was to examine how antigen retrieval affected the yield of immunogold labeling on epoxy sections based on embedding with different amounts of accelerator. The concentration of accelerator DMP-30 (tri(dimethyl amino methyl) phenol) was varied in the range of 0-8% in the processing of the tissue for epoxy embedding. Immunogold labeling was performed on epoxy sections and LR-White sections of fibrin clots and renal tissue with IgG-deposits, and the antibodies used were anti-fibrinogen anti-IgG and, respectively. For some of the sections antigen retrieval was performed by heating the sections in citrate buffer. In all cases, the yield of immunogold labeling increased following antigen retrieval. The increase (%) in the yield of immunogold labeling as a result of antigen retrieval was larger for epoxy sections than for LR-White sections. The immunolabeling on high-accelerator epoxy sections exposed to antigen retrieval was about 20% more intense than on untreated LR-White sections both for IgG and fibrinogen. In addition to breaking fixations bonds introduced by the chemical fixation, we believe that the antigen retrieval also breaks bonds between the epoxy resin and the embedded tissue. The combination of increased amount of accelerator during tissue processing for epoxy embedding and antigen retrieval by heating in citrate buffer is a potent method for increasing specific immunolabeling on epoxy sections.

Acrylic Resins↗

PubMedDate not supplied

A new immunoelectron microscopy approach for the detection of immunoglobulin and complement deposits in epoxy-embedded renal biopsies.

The purpose of this study was to examine the diagnostic value of a new immunoelectron microscopy technique (IEM) for detection of immunoglobulin and complement deposits in epoxy-embedded renal biopsies. Twenty-four renal biopsies were embedded in epoxy resin following a tissue processing involving moderately increased amount of accelerator, DMP-30 (Tri(Dimethyl Amino Methyl) Phenol), in the infiltration steps. Following antigen retrieval by heating in citrate buffer, immunogold labeling was performed on ultrathin sections from these epoxy blocks with antibodies against immunoglobulins and complement. The sections were counterstained with urnayl acetate and lead citrate without any enhancing procedures. The preservation of the ultrastructure with this method was similar to that usually seen in epoxy embedded material. The immunogold labeling was intense and distinct. Immunofluorescence (IF) for light microscopy was carried out on frozen sections of parallel tissue samples. The correspondence between IF and IEM were good, but in some cases higher sensitivity for IgA with IEM than IF was observed in the sense that smaller amounts of antigen were detectable with IEM. The combination of moderately increased amount of accelerator and antigen retrieval is superior to previous methods with respect to ease of use, ultrastructural preservation, and intensity of the immunolabeling. Moreover, the renal tissue can be processed in an automatic ultraprocessor together with other specimens which are to be prepared for routine electron microscopy.

Antigen-Antibody Complex↗