title: Immunolocalization of multiple membrane proteins on a carbon replica with STEM and EDX.
abstract: We present a method for immunolabeling of multiple species of membrane proteins with high spatial resolution. It allows differentiation of equally sized very small markers with different chemical compositions, which leads to high labeling efficiency and reduces steric hindrance of closely spaced immunolabeled biomolecules. Markers such as CdSe/ZnS semiconductor quantum dots and colloidal gold particles are distinguished by differential contrast in high-angle annular detector dark-field STEM mode or by EDX microanalysis of their elemental contents. This method was tested by observation of labeled AMPA- and NMDA-type glutamate receptors on sodium-dodecyl-sulfate-digested replica prepared from rat hippocampus. To improve particle visibility and detectability, the replica films were made exclusively with carbon to avoid the high background of conventional platinum/carbon replica. Extension of the method is suggested by detection of 1.4 nm nanogold particles and its potential application in the biological imaging research.
authors.affiliation: School of Life Science, The Graduate University for Advanced Studies Sokendai, Okazaki 444-8787, Japan. alexandre_loukanov@yahoo.com
authors.forename: Alexandre
authors.initials: A
authors.lastname: Loukanov
authors.forename: Naomi
authors.initials: N
authors.lastname: Kamasawa
authors.forename: Radostin
authors.initials: R
authors.lastname: Danev
authors.forename: Ryuichi
authors.initials: R
authors.lastname: Shigemoto
authors.forename: Kuniaki
authors.initials: K
authors.lastname: Nagayama
mesh_terms: D000818:Animals; D002244:Carbon; D002462:Cell Membrane / Q000378:metabolism; D005614:Freeze Fracturing / Q000379:methods*; D017979:Gold Colloid; D006624:Hippocampus / Q000166:cytology; D007150:Immunohistochemistry / Q000379:methods*; D008565:Membrane Proteins / Q000378:metabolism*; D008855:Microscopy, Electron, Scanning / Q000379:methods*; D016202:N-Methylaspartate / Q000378:metabolism; D053758:Nanoparticles; D051381:Rats; D017470:Receptors, Glutamate / Q000378:metabolism; D013052:Spectrometry, X-Ray Emission / Q000379:methods*; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000378:metabolism
chemical_list: D017979:Gold Colloid; D008565:Membrane Proteins; D017470:Receptors, Glutamate; D016202:N-Methylaspartate; D002244:Carbon; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid