ENHANCEMENT OF IMMUNOGOLD-LABELLED FOCAL ADHESION SITES IN FIBROBLASTS CULTURED ON METAL SUBSTRATES: PROBLEMS AND SOLUTIONS
Visualisation of cell adhesion patterns by scanning electron microscopy requires special preparation and labelling. The membranes and cytoplasm must be removed, without damaging the antigen, to facilitate antibody access to vinculin in the focal adhesions. Low beam energy imaging is used to visualis...
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| Veröffentlicht in: | Cell biology international 2001-12, Vol.25 (12), p.1251-1259 |
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| creator | Owen, G.Rh Meredith, D.O. Ap Gwynn, I. Richards, R.G. |
| description | Visualisation of cell adhesion patterns by scanning electron microscopy requires special preparation and labelling. The membranes and cytoplasm must be removed, without damaging the antigen, to facilitate antibody access to vinculin in the focal adhesions. Low beam energy imaging is used to visualise the cell undersurface (embedded in resin after staining with osmium tetroxide) and immunogold-labelled adhesion sites. The gold probe, must be large enough (>40nm) for detection, while viewing the whole cell, but large gold markers increase steric hindrance and decrease labelling efficiency. This problem can be overcome by using small gold probes (1–5nm) followed by enlargement with silver enhancement, but osmium tetroxide stain etches the silver. We demonstrated that metal substrates increased this etching. Reducing the concentration of osmium tetroxide and incubation time reduced the amount of etching. We have demonstrated that gold enhancement was not etched by osmium tetroxide, irrespective of the substrate. Therefore, comparative studies of cell adhesion to different biomaterial substrates can be performed using immunogold-labelling with gold enhancement. |
| doi_str_mv | 10.1006/cbir.2001.0846 |
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The membranes and cytoplasm must be removed, without damaging the antigen, to facilitate antibody access to vinculin in the focal adhesions. Low beam energy imaging is used to visualise the cell undersurface (embedded in resin after staining with osmium tetroxide) and immunogold-labelled adhesion sites. The gold probe, must be large enough (>40nm) for detection, while viewing the whole cell, but large gold markers increase steric hindrance and decrease labelling efficiency. This problem can be overcome by using small gold probes (1–5nm) followed by enlargement with silver enhancement, but osmium tetroxide stain etches the silver. We demonstrated that metal substrates increased this etching. Reducing the concentration of osmium tetroxide and incubation time reduced the amount of etching. We have demonstrated that gold enhancement was not etched by osmium tetroxide, irrespective of the substrate. Therefore, comparative studies of cell adhesion to different biomaterial substrates can be performed using immunogold-labelling with gold enhancement.</description><identifier>ISSN: 1065-6995</identifier><identifier>EISSN: 1095-8355</identifier><identifier>DOI: 10.1006/cbir.2001.0846</identifier><identifier>PMID: 11748918</identifier><language>eng</language><publisher>Oxford, UK: Elsevier Ltd</publisher><subject>Acrylic Resins ; Animals ; Cell Adhesion ; Cell Membrane - ultrastructure ; Cells, Cultured ; Fibroblasts - physiology ; Fibroblasts - ultrastructure ; Focal Adhesions ; Gold - chemistry ; gold enhancement ; immunogold ; Immunohistochemistry ; Mice ; Mice, Inbred BALB C ; Microscopy, Electron, Scanning - methods ; Microscopy, Immunoelectron - methods ; Osmium Tetroxide - chemistry ; Plastic Embedding ; SEM ; Silver - chemistry ; silver enhancement ; Stainless Steel - chemistry ; Vinculin - metabolism</subject><ispartof>Cell biology international, 2001-12, Vol.25 (12), p.1251-1259</ispartof><rights>2001 Academic Press</rights><rights>The Author(s) Journal compilation © 2001 International Federation for Cell Biology</rights><rights>Copyright 2001 Academic Press.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c4232-600888fb013377701d25fbe176fc22851d8b9f223294c94b79b7f5d93660cea3</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1006%2Fcbir.2001.0846$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1006%2Fcbir.2001.0846$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>314,776,780,1411,27901,27902,45550,45551</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/11748918$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Owen, G.Rh</creatorcontrib><creatorcontrib>Meredith, D.O.</creatorcontrib><creatorcontrib>Ap Gwynn, I.</creatorcontrib><creatorcontrib>Richards, R.G.</creatorcontrib><title>ENHANCEMENT OF IMMUNOGOLD-LABELLED FOCAL ADHESION SITES IN FIBROBLASTS CULTURED ON METAL SUBSTRATES: PROBLEMS AND SOLUTIONS</title><title>Cell biology international</title><addtitle>Cell Biol Int</addtitle><description>Visualisation of cell adhesion patterns by scanning electron microscopy requires special preparation and labelling. The membranes and cytoplasm must be removed, without damaging the antigen, to facilitate antibody access to vinculin in the focal adhesions. Low beam energy imaging is used to visualise the cell undersurface (embedded in resin after staining with osmium tetroxide) and immunogold-labelled adhesion sites. The gold probe, must be large enough (>40nm) for detection, while viewing the whole cell, but large gold markers increase steric hindrance and decrease labelling efficiency. This problem can be overcome by using small gold probes (1–5nm) followed by enlargement with silver enhancement, but osmium tetroxide stain etches the silver. We demonstrated that metal substrates increased this etching. Reducing the concentration of osmium tetroxide and incubation time reduced the amount of etching. We have demonstrated that gold enhancement was not etched by osmium tetroxide, irrespective of the substrate. Therefore, comparative studies of cell adhesion to different biomaterial substrates can be performed using immunogold-labelling with gold enhancement.</description><subject>Acrylic Resins</subject><subject>Animals</subject><subject>Cell Adhesion</subject><subject>Cell Membrane - ultrastructure</subject><subject>Cells, Cultured</subject><subject>Fibroblasts - physiology</subject><subject>Fibroblasts - ultrastructure</subject><subject>Focal Adhesions</subject><subject>Gold - chemistry</subject><subject>gold enhancement</subject><subject>immunogold</subject><subject>Immunohistochemistry</subject><subject>Mice</subject><subject>Mice, Inbred BALB C</subject><subject>Microscopy, Electron, Scanning - methods</subject><subject>Microscopy, Immunoelectron - methods</subject><subject>Osmium Tetroxide - chemistry</subject><subject>Plastic Embedding</subject><subject>SEM</subject><subject>Silver - chemistry</subject><subject>silver enhancement</subject><subject>Stainless Steel - chemistry</subject><subject>Vinculin - metabolism</subject><issn>1065-6995</issn><issn>1095-8355</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2001</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqF0M1v0zAYBnALgdgoXDkin7ils_NhO9yS1G0jOclUJ-JoNY4jBdp1i1tg4p_HUapxQpzsw-997PcB4CNGS4wQudPtMC59hPASsZC8ArcYxZHHgih6Pd1J5JE4jm7AO2u_OYVDRt6CG4xpyGLMbsFvXm6TMuMFL2tYrWFeFE1ZbSqx8kSSciH4Cq6rLBEwWW25zKsSyrzmEuYlXOfprkpFImsJs0bUzc5hBwpeOy-bVNa7xNkv8H5yvJAwKVdQVqKpXZB8D970-4M1H67nAtRrXmdbT1Sb3D3p6dAPfI8gxBjrW4SDgFKKcOdHfWswJb32fRbhjrVx7zsahzoOWxq3tI-6OCAEabMPFuDzHPs4np4uxp7VcbDaHA77B3O6WEX9gCJGmIPLGerxZO1oevU4Dsf9-KwwUlPbampbTW2rqW038OmafGmPpvvLr_U6EM7g53Awz_-JU1malxi57yyAN48N9mx-vYztx--K0IBG6mu5UVtMxT3DoRLOs9kb1-KPwYzK6sE8aNMNo9Fn1Z2Gf63wB-dQogM</recordid><startdate>200112</startdate><enddate>200112</enddate><creator>Owen, G.Rh</creator><creator>Meredith, D.O.</creator><creator>Ap Gwynn, I.</creator><creator>Richards, R.G.</creator><general>Elsevier Ltd</general><general>Blackwell Publishing Ltd</general><scope>BSCLL</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>200112</creationdate><title>ENHANCEMENT OF IMMUNOGOLD-LABELLED FOCAL ADHESION SITES IN FIBROBLASTS CULTURED ON METAL SUBSTRATES: PROBLEMS AND SOLUTIONS</title><author>Owen, G.Rh ; Meredith, D.O. ; Ap Gwynn, I. ; Richards, R.G.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4232-600888fb013377701d25fbe176fc22851d8b9f223294c94b79b7f5d93660cea3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2001</creationdate><topic>Acrylic Resins</topic><topic>Animals</topic><topic>Cell Adhesion</topic><topic>Cell Membrane - ultrastructure</topic><topic>Cells, Cultured</topic><topic>Fibroblasts - physiology</topic><topic>Fibroblasts - ultrastructure</topic><topic>Focal Adhesions</topic><topic>Gold - chemistry</topic><topic>gold enhancement</topic><topic>immunogold</topic><topic>Immunohistochemistry</topic><topic>Mice</topic><topic>Mice, Inbred BALB C</topic><topic>Microscopy, Electron, Scanning - methods</topic><topic>Microscopy, Immunoelectron - methods</topic><topic>Osmium Tetroxide - chemistry</topic><topic>Plastic Embedding</topic><topic>SEM</topic><topic>Silver - chemistry</topic><topic>silver enhancement</topic><topic>Stainless Steel - chemistry</topic><topic>Vinculin - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Owen, G.Rh</creatorcontrib><creatorcontrib>Meredith, D.O.</creatorcontrib><creatorcontrib>Ap Gwynn, I.</creatorcontrib><creatorcontrib>Richards, R.G.</creatorcontrib><collection>Istex</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Cell biology international</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Owen, G.Rh</au><au>Meredith, D.O.</au><au>Ap Gwynn, I.</au><au>Richards, R.G.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>ENHANCEMENT OF IMMUNOGOLD-LABELLED FOCAL ADHESION SITES IN FIBROBLASTS CULTURED ON METAL SUBSTRATES: PROBLEMS AND SOLUTIONS</atitle><jtitle>Cell biology international</jtitle><addtitle>Cell Biol Int</addtitle><date>2001-12</date><risdate>2001</risdate><volume>25</volume><issue>12</issue><spage>1251</spage><epage>1259</epage><pages>1251-1259</pages><issn>1065-6995</issn><eissn>1095-8355</eissn><abstract>Visualisation of cell adhesion patterns by scanning electron microscopy requires special preparation and labelling. The membranes and cytoplasm must be removed, without damaging the antigen, to facilitate antibody access to vinculin in the focal adhesions. Low beam energy imaging is used to visualise the cell undersurface (embedded in resin after staining with osmium tetroxide) and immunogold-labelled adhesion sites. The gold probe, must be large enough (>40nm) for detection, while viewing the whole cell, but large gold markers increase steric hindrance and decrease labelling efficiency. This problem can be overcome by using small gold probes (1–5nm) followed by enlargement with silver enhancement, but osmium tetroxide stain etches the silver. We demonstrated that metal substrates increased this etching. Reducing the concentration of osmium tetroxide and incubation time reduced the amount of etching. We have demonstrated that gold enhancement was not etched by osmium tetroxide, irrespective of the substrate. Therefore, comparative studies of cell adhesion to different biomaterial substrates can be performed using immunogold-labelling with gold enhancement.</abstract><cop>Oxford, UK</cop><pub>Elsevier Ltd</pub><pmid>11748918</pmid><doi>10.1006/cbir.2001.0846</doi><tpages>9</tpages></addata></record> |
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| subjects | Acrylic Resins Animals Cell Adhesion Cell Membrane - ultrastructure Cells, Cultured Fibroblasts - physiology Fibroblasts - ultrastructure Focal Adhesions Gold - chemistry gold enhancement immunogold Immunohistochemistry Mice Mice, Inbred BALB C Microscopy, Electron, Scanning - methods Microscopy, Immunoelectron - methods Osmium Tetroxide - chemistry Plastic Embedding SEM Silver - chemistry silver enhancement Stainless Steel - chemistry Vinculin - metabolism |
| title | ENHANCEMENT OF IMMUNOGOLD-LABELLED FOCAL ADHESION SITES IN FIBROBLASTS CULTURED ON METAL SUBSTRATES: PROBLEMS AND SOLUTIONS |
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