Two approaches to double post-embedding immunogold labeling of freeze-substituted tissue embedded in low temperature Lowicryl HM20 resin.
Double labeling is used for localizing two antigens simultaneously in the same tissue. We have used two approaches to post-embedding immunogold labeling to investigate whether nerve terminals in the guinea-pig anteroventral cochlear nucleus (AVCN) that contain gamma-aminobutyric acid (GABA) or glycine are capable of retrieving the other amino acid as part of an investigation of colocalization of these putative neurotransmitters. For this, vibroslices of perfusion-fixed brain stem were freeze-substituted and embedded in the low temperature resin, Lowicryl HM20. Simultaneous labeling of ultrathin sections was then performed with a mixture of a rabbit primary antibody to GABA and a guinea-pig primary antibody to the glycine transporter, GLYT2, followed by labeling with a mixture of secondary antibodies (goat anti-rabbit IgG-30 nm gold, goat anti-guinea pig IgG-15 nm gold). This approach indicated that GLYT2 occurs in the plasma membrane of some terminals that contain GABA. The other approach involved sequential labeling of ultrathin sections with a rabbit primary antibody to the GABA transporter, GAT1, followed by an anti-rabbit secondary antibody conjugated to 15-nm gold particles. Sections were then treated with paraformaldehyde vapor to denature any free anti-IgG binding sites on the first antibody, and labeled with a primary antibody to glycine also raised in rabbit followed by an anti-rabbit secondary antibody conjugated to 30-nm gold particles. This approach indicated that GAT1 occurs in the plasma membrane of some terminals that contain glycine. Thus, these techniques can be used to localize heat-labile multiple antigens in the same tissue.
MacKay Institute of Communication and Neuroscience, School of Life Sciences, Keele University, Keele, Staffordshire ST5 5BG, UK.
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Mahendrasingam
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Wallam
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Hackney
D000180:Acrylic Resins; D026921:Amino Acid Transport Systems, Neutral / Q000276:immunology / Q000378:metabolism; D000818:Animals; D000906:Antibodies; D001665:Binding Sites / Q000276:immunology; D002352:Carrier Proteins / Q000276:immunology / Q000378:metabolism; D002462:Cell Membrane / Q000276:immunology / Q000378:metabolism; D017626:Cochlear Nucleus / Q000378:metabolism / Q000648:ultrastructure; D005260:Female; D017110:Freeze Substitution / Q000379:methods*; D050485:GABA Plasma Membrane Transport Proteins; D005998:Glycine / Q000276:immunology / Q000378:metabolism; D050487:Glycine Plasma Membrane Transport Proteins; D006168:Guinea Pigs; D007150:Immunohistochemistry / Q000379:methods*; D008297:Male; D008565:Membrane Proteins / Q000276:immunology / Q000378:metabolism; D026901:Membrane Transport Proteins; D008854:Microscopy, Electron / Q000295:instrumentation / Q000379:methods*; D008867:Microtomy / Q000295:instrumentation / Q000379:methods*; D027361:Organic Anion Transporters; D017729:Presynaptic Terminals / Q000378:metabolism / Q000648:ultrastructure; D016610:Tissue Embedding / Q000379:methods*; D005680:gamma-Aminobutyric Acid / Q000276:immunology / Q000378:metabolism
D000180:Acrylic Resins; D026921:Amino Acid Transport Systems, Neutral; D000906:Antibodies; D002352:Carrier Proteins; D050485:GABA Plasma Membrane Transport Proteins; D050487:Glycine Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D027361:Organic Anion Transporters; D005680:gamma-Aminobutyric Acid; D005998:Glycine