title: Post-embedding Mammalian Tissue for Immunoelectron Microscopy: A Standardized Procedure Based on Heat-Induced Antigen Retrieval.
abstract: We describe a standardized method of fixation, antigen retrieval, and image contrasting for post-embedding immunoelectron microscopy. Tissues are fixed with formaldehyde solutions containing Ca(2+) and Mg(2+) ions at pH 7.4 and then at pH 8.5. After dehydration with dimethylformamide, the specimens are embedded in LR-White resin. For antigen retrieval, ultrathin sections are heated in 0.5 M Tris-HCl, pH 9.0, for 1-2 h at 95 °C. After immunogold labeling, the sections are treated with a mixture of tannic acid and glutaraldehyde, with OsO4 solution, and then double-stained with uranyl acetate and lead citrate. The standardized method yields strong and reproducible immunoreactions for many antigens showing excellent image contrast without destruction of fine structures.
authors: Department of Pathology, School of Medicine, Keio University, 35-Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan. shuji@z5.keio.jp.
authors: Shuji
authors: S
authors: Yamashita
mesh_terms: D000180:Acrylic Resins / Q000737:chemistry; D000818:Animals; D000906:Antibodies / Q000737:chemistry; D000941:Antigens / Q000235:genetics* / Q000378:metabolism; D003890:Desiccation / Q000379:methods; D004126:Dimethylformamide / Q000737:chemistry; D004721:Endoplasmic Reticulum / Q000378:metabolism / Q000648:ultrastructure; D005404:Fixatives / Q000737:chemistry; D005557:Formaldehyde / Q000737:chemistry; D015870:Gene Expression; D017979:Gold Colloid / Q000737:chemistry*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry / Q000379:methods*; D007668:Kidney / Q000378:metabolism / Q000648:ultrastructure; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D016253:Microscopy, Immunoelectron / Q000379:methods*; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*; D013634:Tannins / Q000737:chemistry; D016610:Tissue Embedding / Q000379:methods; D016707:Tissue Fixation / Q000379:methods*
chemical_list: D000180:Acrylic Resins; D000906:Antibodies; D000941:Antigens; D005404:Fixatives; D017979:Gold Colloid; D026901:Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D011956:Receptors, Cell Surface; D013634:Tannins; C489997:Tomm20 protein, mouse; D005557:Formaldehyde; D004126:Dimethylformamide; C048707:LR white
keywords: Electron staining with tannic acid; Heat-induced antigen retrieval; Immunoelectron microscopy; LR-White; Mammalian tissue; Post-embedding method; Universal fixatives