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Updated: Aug 9, 2026
Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures
Published on: April 3, 2013
Two approaches to double post-embedding immunogold labeling of freeze-substituted tissue embedded in low temperature
S Mahendrasingam1, C A Wallam, C M Hackney
1MacKay Institute of Communication and Neuroscience, School of Life Sciences, Keele University, Keele, Staffordshire ST5 5BG, UK.
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.
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.
