The post-embedding method for immunoelectron microscopy of mammalian tissues: 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 20 mM Tris-HCl buffer (pH 9.0) for 1 h at 95 degrees C. After immunogold labeling, the sections are treated with a mixture of tannic acid and glutaraldehyde, with OsO(4) 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: Shuji Yamashita
• Journal: Methods in molecular biology (Clifton, N.J.) (Methods Mol Biol) Vol. 657 Pg. 237-48 ( 2010) ISSN: 1940-6029 [Electronic] United States
• PMID: 20602221 (Publication Type: Journal Article)

Chemical References

• Antibodies
• Antigens
• Gold Colloid

Topics

• Animals
• Antibodies (immunology)
• Antigens (analysis, immunology)
• Gold Colloid (chemistry, immunology)
• Hot Temperature
• Kidney (cytology, ultrastructure)
• Mice
• Microscopy, Immunoelectron (methods, standards)
• Mitochondrial Membranes (immunology)
• Tissue Embedding (methods, standards)