ULTRASTRUCTURAL CHARACTERIZATION OF NEURONS RECORDED INTRACELLULARLY IN-VIVO AND INJECTED WITH LUCIFER YELLOW - ADVANTAGES OF IMMUNOGOLD-SILVER VSIMMUNOPEROXIDASE LABELING
ULTRASTRUCTURAL CHARACTERIZATION OF NEURONS RECORDED INTRACELLULARLY IN-VIVO AND INJECTED WITH LUCIFER YELLOW - ADVANTAGES OF IMMUNOGOLD-SILVER VSIMMUNOPEROXIDASE LABELING
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DOI：
10.1002/jemt.1070300509
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发表时间：
1995-04-01
影响因子：
2.5
通讯作者：
PICKEL, VM
中科院分区：
文献类型：
--
作者：
BRANCHEREAU, P;VANBOCKSTAELE, EJ;PICKEL, VM
荧光黄的免疫过氧化物酶标记提供了一种敏感的方法，在细胞内记录在体外或体内的神经元的形态特征。然而，反应产物往往是如此密集，它掩盖了必要的突触接触到个别填充神经元的分析超微结构的细节。在本研究中，我们描述了一个银强化程序，使用1 nm的金标记的荧光黄作为一个最佳的手段免疫细胞化学识别单个生理特征的神经元在超微结构水平。麻醉大鼠额叶皮层单个神经元在体内穿刺，并充满荧光黄。然后用丙烯醛固定剂灌注大脑。通过记录位点的单个振动切片机切片与针对荧光黄的兔抗体反应，然后与山羊抗兔1 nm金标记IgG和银增强的抗体反应。为了进行比较，对另外的切片进行处理，用于荧光黄的免疫过氧化物酶检测。处理标记的切片用于光学显微镜检查或包埋在塑料中用于电子显微镜检查。荧光黄的免疫金-银标记以及过氧化物酶反应产物在细胞体、近端和远端树突和棘中很容易检测到。然而，与免疫过氧化物酶相反，免疫金-银反应并不掩盖亚细胞器。最重要的是，突触连接形成的传入填充神经元更容易识别后的免疫金-银程序。这种清晰的突触后密度的可视化对于检查传入神经和生理特征神经元之间的突触回路是必不可少的。(C)Wiiey-Liss，Inc.
Immunoperoxidase labeling of lucifer yellow provides a sensitive method for morphological characterization of neurons recorded intracellularly in vitro or in vivo. However, the reaction product is often so dense that it obscures ultrastructural details necessary for the analysis of synaptic contacts onto individually filled neurons. In the present study, we describe a silver intensification procedure using 1 nm gold labeling of lucifer yellow as an optimal means for immunocytochemically identifying single physiologically characterized neurons at the ultrastructural level. Single neurons in the frontal cortex of anesthetized rats were impaled in vivo and filled with lucifer yellow. The brains were then perfused with an acrolein fixative. Single vibratome sections through the recording site were reacted with a rabbit antibody directed against lucifer yellow followed by goat anti-rabbit 1 nm gold-labeled IgG and silver intensified. For comparison, additional sections were processed for immunoperoxidase detection of lucifer yellow. Labeled sections were processed for light microscopy or embedded in plastic for electron microscopy. The immunogold-silver label as well as peroxidase reaction product of lucifer yellow was readily detected in cell bodies, proximal and distal dendrites, and spines. However, in contrast to immunoperoxidase, the immunogold-silver reaction did not obscure subcellular organelles. Most importantly, the synaptic junctions formed by afferents to the filled neuron were more easily identifiable following the immunogold-silver procedure. This clear visualization of postsynaptic densities is essential for examining synaptic circuitry between afferents and physiologically characterized neurons. (C) 1995 Wiiey-Liss, Inc.