Axons and axon terminals of cerebellar Purkinje cells and basket cells have higher levels of parvalbumin immunoreactivity than somata and dendrites: quantitative analysis by immunogold labeling
Axons and axon terminals of cerebellar Purkinje cells and basket cells have higher levels of parvalbumin immunoreactivity than somata and dendrites: quantitative analysis by immunogold labeling
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小脑浦肯野细胞和篮子细胞的轴突和轴突末端比体细胞和树突具有更高水平的小白蛋白免疫反应性：免疫金标记定量分析
DOI：
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发表时间：
2004
影响因子：
2
通讯作者：
Claus W. Heizmann
中科院分区：
文献类型：
--
作者：
T. Kosaka;K. Kosaka;T. Nakayama;Willi Hunziker;Claus W. Heizmann
钙结合蛋白parvalbumin（PV）和calbindin D28 K的免疫强度定量在不同部位的浦肯野细胞和中间神经元（篮状细胞和星状细胞）的大鼠小脑。在Lowicryl K4 M包埋的薄切片上应用了电子显微镜、接种后免疫金程序。使用两种一抗（小鼠单克隆抗大鼠钙结合蛋白D28 K和兔多克隆抗大鼠PV）的组合，通过双标记免疫细胞化学鉴定神经元概况。将二抗与不同尺寸（10和15 nm直径）的胶体金缀合。在小脑皮质，双标记的配置文件被确定为浦肯野细胞和配置文件仅与抗PV标记被确定为interneurons。金颗粒的密度用于统计比较浦肯野细胞的胞体、树突、树突棘、轴突和轴突终末以及中间神经元中PV和钙结合蛋白D28 K的相对水平。浦肯野细胞和篮状细胞的轴突和轴突终末的PV免疫反应性水平显着高于浦肯野细胞胞体，初级，二级和三级树突，树突棘，以及中间神经元胞体。另一方面，本研究不能确定钙结合蛋白D28 K是否均匀分布在浦肯野细胞的索马、树突和轴突中，或者也集中在浦肯野细胞轴突中。为了估计PV的绝对浓度，我们制备了一系列人工标准样品，这些样品是含有给定浓度PV（0、12.5、25、50、100、200和400 μM，1和2 mM）的固定的10%牛血清白蛋白，并从这些标准样品的定量免疫金分析中推导出校准曲线。我们还分析了一个快速收缩的肌肉，腓肠肌（GCM），其PV含量的浅表部分先前在生化研究中报告; GCM和标准样品的金颗粒密度之间的比较表明，这些人工标准样品可用于估计PV的近似细胞内浓度。根据这些分析，浦肯野细胞胞体和树突以及中间神经元胞体中的PV浓度估计为50-100 μM，浦肯野细胞和篮状细胞的轴突和轴突终末中的PV浓度估计为1 mM或更高。
The immunointensities of calcium-binding proteins parvalbumin (PV) and calbindin D28K were quantified in different parts of Purkinje cells and interneurons (basket cells and stellate cells) of the rat cerebellum. An electron microscopic, postembedding immunogold procedure on Lowicryl K4M-embedded thin sections was applied. Neuronal profiles were identified by double-labeling immunocytochemistry using the combination of the two primary antibodies, mouse monoclonal anti-rat calbindin D28K and rabbit polyclonal anti-rat PV. The secondary antibodies were conjugated with colloidal gold of different sizes (10 and 15 nm diameter). In the cerebellar cortex, double-labeled profiles were identified as Purkinje cells and profiles labeled only with anti-PV were identified as inteneurons. The densities of gold particles were used for statistical comparison of the relative levels of PV and calbindin D28K in somata, dendrites, dendritic spines, axons and axon terminals of Purkinje cells, and interneurons. The axons and axon terminals of Purkinje cells and basket cells had significantly higher levels of PV immunoreactivity than Purkinje cell somata, primary, secondary, and tertiary dendrites, and dendritic spines, as well as interneuron somata. On the other hand, the present study could not determine conclusively whether calbindin D28K was distributed homogeneously throughout soma, dendrites, and axons of Purkinje cells or was also concentrated in Purkinje cell axons. To estimate absolute PV concentrations, we made a series of artificial standard samples which were aldehyde-fixed 10% bovine serum albumin containing given concentrations of PV (0, 12.5, 25, 50, 100, 200, and 400 μM, 1 and 2 mM), and calibration curves were deduced from quantitative immunogold analyses of these standard samples. We also analyzed a fast twitch muscle, the superficial part of the gastrocnemius muscle (GCM), whose PV content was previously reported in a biochemical study; the comparison between gold particle densities of GCM and standard samples indicated that these artificial standard samples could be used to estimate the approximate intracellular concentrations of PV. Based on these analyses PV concentrations were estimated as 50-100 μM in Purkinje cell somata and dendrites as well as interneuron somata, and as 1 mM or more in axons and axon terminals of Purkinje cells and basket cells.
用于免疫金电子显微镜的脑组织 Lowicryl K4M 包埋。
DOI：
10.1177/33.9.2991364
发表时间：
1985
期刊：
The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society
影响因子：
--
作者：
Valentino,KL;Crumrine,DA;Reichardt,LF
通讯作者：
Reichardt,LF