Immunogold Labeling

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Thomas Yasumura - One of the best experts on this subject based on the ideXlab platform.

  • connexin composition in apposed gap junction hemiplaques revealed by matched double replica freeze fracture replica Immunogold Labeling
    The Journal of Membrane Biology, 2012
    Co-Authors: John E Rash, Naomi Kamasawa, Kimberly G V Davidson, Thomas Yasumura, Alberto E Pereda, J I Nagy
    Abstract:

    Despite the combination of light-microscopic immunocytochemistry, histochemical mRNA detection techniques and protein reporter systems, progress in identifying the protein composition of neuronal versus glial gap junctions, determination of the differential localization of their constituent connexin proteins in two apposing membranes and understanding human neurological diseases caused by connexin mutations has been problematic due to ambiguities introduced in the cellular and subcellular assignment of connexins. Misassignments occurred primarily because membranes and their constituent proteins are below the limit of resolution of light microscopic imaging techniques. Currently, only serial thin-section transmission electron microscopy and freeze-fracture replica Immunogold Labeling have sufficient resolution to assign connexin proteins to either or both sides of gap junction plaques. However, freeze-fracture replica Immunogold Labeling has been limited because conventional freeze fracturing allows retrieval of only one of the two membrane fracture faces within a gap junction, making it difficult to identify connexin coupling partners in hemiplaques removed by fracturing. We now summarize progress in ascertaining the connexin composition of two coupled hemiplaques using matched double-replicas that are labeled simultaneously for multiple connexins. This approach allows unambiguous identification of connexins and determination of the membrane “sidedness” and the identities of connexin coupling partners in homotypic and heterotypic gap junctions of vertebrate neurons.

  • gap junctions on hippocampal mossy fiber axons demonstrated by thin section electron microscopy and freeze fracture replica Immunogold Labeling
    Proceedings of the National Academy of Sciences of the United States of America, 2007
    Co-Authors: Farid Hamzeisichani, Naomi Kamasawa, Kimberly G V Davidson, Thomas Yasumura, William G M Janssen, Patrick R Hof, Susan L Wearne, Mark Stewart, Steven R Young, Miles A Whittington
    Abstract:

    Gap junctions have been postulated to exist between the axons of excitatory cortical neurons based on electrophysiological, modeling, and dye-coupling data. Here, we provide ultrastructural evidence for axoaxonic gap junctions in dentate granule cells. Using combined confocal laser scanning microscopy, thin-section transmission electron microscopy, and grid-mapped freeze–fracture replica Immunogold Labeling, 10 close appositions revealing axoaxonic gap junctions (≈30–70 nm in diameter) were found between pairs of mossy fiber axons (≈100–200 nm in diameter) in the stratum lucidum of the CA3b field of the rat ventral hippocampus, and one axonal gap junction (≈100 connexons) was found on a mossy fiber axon in the CA3c field of the rat dorsal hippocampus. Immunogold Labeling with two sizes of gold beads revealed that connexin36 was present in that axonal gap junction. These ultrastructural data support computer modeling and in vitro electrophysiological data suggesting that axoaxonic gap junctions play an important role in the generation of very fast (>70 Hz) network oscillations and in the hypersynchronous electrical activity of epilepsy.

  • direct Immunogold Labeling of connexins and aquaporin 4 in freeze fracture replicas of liver brain and spinal cord factors limiting quantitative analysis
    Cell and Tissue Research, 1999
    Co-Authors: John E Rash, Thomas Yasumura
    Abstract:

    Direct Immunogold Labeling and histological mapping of membrane proteins is demonstrated in Lexan-stabilized SDS-washed freeze-fracture replicas of complex tissues. Using rat brain and spinal cord as primary model systems and liver as a ”control” tissue to identify preparation and Labeling artifacts, we demonstrate the presence of connexin43 in freeze-fractured gap junctions of identified and mapped astrocytes and ependymocytes, and confirm the presence of connexin32 in freeze-fractured gap junctions in liver. In addition, the simultaneous double-Labeling of dissimilar proteins (connexin43 and aquaporin-4) is demonstrated in gap junctions and square arrays, respectively, in the plasma membranes of astrocytes and ependymocytes. Finally, double-side shadowing and conventional staining methods are used to reveal the extent of biological material present at the time of Labeling and to investigate the dynamics of membrane solubilization, the primary artifacts that occur during Labeling, and several factors limiting quantitative analysis.

  • direct Immunogold Labeling of aquaporin 4 in square arrays of astrocyte and ependymocyte plasma membranes in rat brain and spinal cord
    Proceedings of the National Academy of Sciences of the United States of America, 1998
    Co-Authors: John E Rash, Thomas Yasumura, C S Hudson, Peter Agre, Soren Nielsen
    Abstract:

    Aquaporin (AQP) water channels are abundant in the brain and spinal cord, where AQP1 and AQP4 are believed to play major roles in water metabolism and osmoregulation. Immunocytochemical analysis of the brain recently revealed that AQP4 has a highly polarized distribution, with marked expression in astrocyte end-feet that surround capillaries and form the glia limitans; however, the structural organization of AQP4 has remained unknown. In freeze-fracture replicas, astrocyte end-feet contain abundant square arrays of intramembrane particles that parallel the distribution of AQP4. To determine whether astrocyte and ependymocyte square arrays contain AQP4, we employed Immunogold Labeling of SDS-washed freeze-fracture replicas and stereoscopic confirmation of tissue binding. Antibodies to AQP4 directly labeled ≈33% of square arrays in astrocyte and ependymocyte plasma membranes in rat brain and spinal cord. Overall, 84% of labels were present beneath square arrays; 11% were beneath particle clusters that resembled square arrays that had been altered during fixation or cleaving; and 5% were beneath the much larger areas of glial plasma membrane that were devoid of square arrays. Based on this evidence that AQP4 is concentrated in glial square arrays, freeze-fracture methods may now provide biophysical insights regarding neuropathological states in which abnormal fluid shifts are accompanied by alterations in the aggregation state or the molecular architecture of square arrays.

John E Rash - One of the best experts on this subject based on the ideXlab platform.

  • connexin composition in apposed gap junction hemiplaques revealed by matched double replica freeze fracture replica Immunogold Labeling
    The Journal of Membrane Biology, 2012
    Co-Authors: John E Rash, Naomi Kamasawa, Kimberly G V Davidson, Thomas Yasumura, Alberto E Pereda, J I Nagy
    Abstract:

    Despite the combination of light-microscopic immunocytochemistry, histochemical mRNA detection techniques and protein reporter systems, progress in identifying the protein composition of neuronal versus glial gap junctions, determination of the differential localization of their constituent connexin proteins in two apposing membranes and understanding human neurological diseases caused by connexin mutations has been problematic due to ambiguities introduced in the cellular and subcellular assignment of connexins. Misassignments occurred primarily because membranes and their constituent proteins are below the limit of resolution of light microscopic imaging techniques. Currently, only serial thin-section transmission electron microscopy and freeze-fracture replica Immunogold Labeling have sufficient resolution to assign connexin proteins to either or both sides of gap junction plaques. However, freeze-fracture replica Immunogold Labeling has been limited because conventional freeze fracturing allows retrieval of only one of the two membrane fracture faces within a gap junction, making it difficult to identify connexin coupling partners in hemiplaques removed by fracturing. We now summarize progress in ascertaining the connexin composition of two coupled hemiplaques using matched double-replicas that are labeled simultaneously for multiple connexins. This approach allows unambiguous identification of connexins and determination of the membrane “sidedness” and the identities of connexin coupling partners in homotypic and heterotypic gap junctions of vertebrate neurons.

  • direct Immunogold Labeling of connexins and aquaporin 4 in freeze fracture replicas of liver brain and spinal cord factors limiting quantitative analysis
    Cell and Tissue Research, 1999
    Co-Authors: John E Rash, Thomas Yasumura
    Abstract:

    Direct Immunogold Labeling and histological mapping of membrane proteins is demonstrated in Lexan-stabilized SDS-washed freeze-fracture replicas of complex tissues. Using rat brain and spinal cord as primary model systems and liver as a ”control” tissue to identify preparation and Labeling artifacts, we demonstrate the presence of connexin43 in freeze-fractured gap junctions of identified and mapped astrocytes and ependymocytes, and confirm the presence of connexin32 in freeze-fractured gap junctions in liver. In addition, the simultaneous double-Labeling of dissimilar proteins (connexin43 and aquaporin-4) is demonstrated in gap junctions and square arrays, respectively, in the plasma membranes of astrocytes and ependymocytes. Finally, double-side shadowing and conventional staining methods are used to reveal the extent of biological material present at the time of Labeling and to investigate the dynamics of membrane solubilization, the primary artifacts that occur during Labeling, and several factors limiting quantitative analysis.

  • direct Immunogold Labeling of aquaporin 4 in square arrays of astrocyte and ependymocyte plasma membranes in rat brain and spinal cord
    Proceedings of the National Academy of Sciences of the United States of America, 1998
    Co-Authors: John E Rash, Thomas Yasumura, C S Hudson, Peter Agre, Soren Nielsen
    Abstract:

    Aquaporin (AQP) water channels are abundant in the brain and spinal cord, where AQP1 and AQP4 are believed to play major roles in water metabolism and osmoregulation. Immunocytochemical analysis of the brain recently revealed that AQP4 has a highly polarized distribution, with marked expression in astrocyte end-feet that surround capillaries and form the glia limitans; however, the structural organization of AQP4 has remained unknown. In freeze-fracture replicas, astrocyte end-feet contain abundant square arrays of intramembrane particles that parallel the distribution of AQP4. To determine whether astrocyte and ependymocyte square arrays contain AQP4, we employed Immunogold Labeling of SDS-washed freeze-fracture replicas and stereoscopic confirmation of tissue binding. Antibodies to AQP4 directly labeled ≈33% of square arrays in astrocyte and ependymocyte plasma membranes in rat brain and spinal cord. Overall, 84% of labels were present beneath square arrays; 11% were beneath particle clusters that resembled square arrays that had been altered during fixation or cleaving; and 5% were beneath the much larger areas of glial plasma membrane that were devoid of square arrays. Based on this evidence that AQP4 is concentrated in glial square arrays, freeze-fracture methods may now provide biophysical insights regarding neuropathological states in which abnormal fluid shifts are accompanied by alterations in the aggregation state or the molecular architecture of square arrays.

Sverrehenning Brorson - One of the best experts on this subject based on the ideXlab platform.

  • the intensity of Immunogold Labeling of deplasticized acrylic sections compared to deplasticized epoxy sections theoretical deductions and experimental data
    Micron, 2008
    Co-Authors: Sverrehenning Brorson, Finn P Reinholt
    Abstract:

    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.

  • heat induced retrieval of Immunogold Labeling for nucleobindin and osteoadherin from lowicryl sections of bone
    Micron, 2006
    Co-Authors: Lene B Solberg, Sverrehenning Brorson, Gunhild Melhus, Mikael Wendel, Finn P Reinholt
    Abstract:

    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.

  • bovine serum albumin bsa as a reagent against non specific Immunogold Labeling on lr white and epoxy resin
    Micron, 1997
    Co-Authors: Sverrehenning Brorson
    Abstract:

    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.

  • 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
    Micron, 1996
    Co-Authors: Sverrehenning Brorson, Fredrik Skjørten
    Abstract:

    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.

  • The theoretical relationship of Immunogold Labeling on acrylic sections and epoxy sections
    Micron (Oxford England : 1993), 1996
    Co-Authors: Sverrehenning Brorson, Fredrik Skjørten
    Abstract:

    Abstract 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.

S.h Brorson - One of the best experts on this subject based on the ideXlab platform.

  • increased level of Immunogold Labeling of epoxy sections by rising the temperature significantly beyond 100 c in the antigen retrieval medium
    Micron, 2001
    Co-Authors: S.h Brorson, G H Nguyen
    Abstract:

    Abstract 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°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°C) than at temperatures just below the normal boiling point. Sections stained with anti-C3 turned from almost negative Labeling when heated at 95°C to strong positive Labeling when heated at 135°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°C. The practical significance of these results is that antigen retrieval of epoxy sections should be performed by heating in aqueous solutions at 135°C or higher to obtain maximum immunoLabeling.

  • Deplasticizing or etching of epoxy sections with different concentrations of sodium ethoxide to enhance the Immunogold Labeling
    Micron (Oxford England : 1993), 2001
    Co-Authors: S.h Brorson
    Abstract:

    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 comparative study of the Immunogold Labeling on H2O2-treated and heated epoxy sections
    Micron (Oxford England : 1993), 2001
    Co-Authors: S.h Brorson, A.r Hansen, H.z Nielsen, I.k Woxen
    Abstract:

    Abstract The purpose of this study was to compare the intensity of the Immunogold Labeling of H2O2-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 H2O2, others were heated in a citrate solution, while some were not treated at all. Some epoxy sections, which had been exposed to both H2O2 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 H2O2-treated sections. The yield of immunoLabeling of the sections that had been exposed to both H2O2 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 H2O2 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 H2O2. We interpret the results from the combined treatment with H2O2 and heat in the following way; the bonds that are broken by H2O2 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 H2O2.

  • antigen detection on resin sections and methods for improving the Immunogold Labeling by manipulating the resin
    Histology and Histopathology, 1998
    Co-Authors: S.h Brorson
    Abstract:

    Considering the importance of immunolocalization of cellular substances combined with good ultrastructure and ease of use, this review is focused on the use of resin and the possibilities of manipulating the resin before and after embedding in order to improve the immunoLabeling of resin sections for electron microscopy. The qualities of acrylic resins and conventional epoxy resin for immunoelectron microscopy are discussed. Acrylic sections are usually more suited for immunoelectron microscopy than conventional epoxy sections. Different etching procedures (sodium ethoxide or sodium metaperiodate) may be applied to conventional epoxy sections to enhance the yield of immunoLabeling. Lately, a method which does not involve any kind of etching has been developed for enhancing the Immunogold Labeling of epoxy sections up to about 8 times. This method involves increased concentration of accelerator in the epoxy resin mixture when processing the tissue. The ultrastructural preservation of the tissue is important in immunoelectron microscopical procedures, and not only the intensity of the immunoLabeling; in this respect no resin may compete with the widely used epoxy resins.

Nicholas J Severs - One of the best experts on this subject based on the ideXlab platform.

  • topography of lipid droplet associated proteins insights from freeze fracture replica Immunogold Labeling
    Journal of Lipids, 2011
    Co-Authors: Horst Robenek, Insa Buers, Oliver Hofnagel, David Troyer, Mirko J Robenek, Anneke Ruebel, Nicholas J Severs
    Abstract:

    Lipid droplets are not merely storage depots for superfluous intracellular lipids in times of hyperlipidemic stress, but metabolically active organelles involved in cellular homeostasis. Our concepts on the metabolic functions of lipid droplets have come from studies on lipid droplet-associated proteins. This realization has made the study of proteins, such as PAT family proteins, caveolins, and several others that are targeted to lipid droplets, an intriguing and rapidly developing area of intensive inquiry. Our existing understanding of the structure, protein organization, and biogenesis of the lipid droplet has relied heavily on microscopical techniques that lack resolution and the ability to preserve native cellular and protein composition. Freeze-fracture replica Immunogold Labeling overcomes these disadvantages and can be used to define at high resolution the precise location of lipid droplet-associated proteins. In this paper illustrative examples of how freeze-fracture immunocytochemistry has contributed to our understanding of the spatial organization in the membrane plane and function of PAT family proteins and caveolin-1 are presented. By revisiting the lipid droplet with freeze-fracture immunocytochemistry, new perspectives have emerged which challenge prevailing concepts of lipid droplet biology and may hopefully provide a timely impulse for many ongoing studies.

  • Recent advances in freeze-fracture electron microscop: the replica immunoLabeling technique
    Biological procedures online, 2008
    Co-Authors: Horst Robenek, Nicholas J Severs
    Abstract:

    Freeze-fracture electron microscopy is a technique for examining the ultrastructure of rapidly frozen biological samples by transmission electron microscopy. Of a range of approaches to freeze-fracture cytochemistry that have been developed and tried the most successful is the technique termed freeze-fracture replica Immunogold Labeling (FRIL). In this technique samples are frozen fractured and replicated with platinum-carbon as in standard freeze fracture and then carefully treated with sodium dodecylsulphate to remove all the biological material except a fine layer of molecules attached to the replica itself. Immunogold Labeling of these molecules permits their distribution to be seen superimposed upon high resolution planar views of membrane structure. Examples of how this technique has contributed to our understanding of lipid droplet biogenesis and function are discussed.

  • co localization of dystrophin and β dystroglycan demonstrated in en face view by double Immunogold Labeling of freeze fractured skeletal muscle
    Journal of Histochemistry and Cytochemistry, 1998
    Co-Authors: Michael J Cullen, John Walsh, Shirley Stevenson, Stephen Rothery, Nicholas J Severs
    Abstract:

    SUMMARY An absence of dystrophin causes Duchenne muscular dystrophy, but the precise mechanism underlying necrosis of the muscle cells is still unclear. Dystrophin and β-dystroglycan are components of a complex of at least nine proteins, the dystrophin-glycoprotein complex (DGC), that links the membrane cytoskeleton to extracellular elements in skeletal and cardiac muscle. Biochemical studies indicate that dystrophin is bound to other components of the DGC via β-dystroglycan, which suggests that the distribution of these two proteins should be almost identical. In this study, therefore, we examined the spatial relationship between dystrophin and β-dystroglycan with a range of different imaging techniques to investigate the extent of the predicted co-localization. We used (a) double Immunogold fracture-label, a freeze-fracture cytochemical technique that allows high-resolution face-on views of labeled membrane components in thin sections and in platinum-carbon replicas, (b) double Immunogold Labeling of cr...