Caceres A, Binder L I, Payne M R, Bender P, Rebhun L, Steward O
J Neurosci. 1984 Feb;4(2):394-410. doi: 10.1523/JNEUROSCI.04-02-00394.1984.
The distribution and subcellular localization of tubulin and MAP2 in brain tissue were analyzed by immunocytochemistry with monoclonal hybridoma antibodies prepared against Chinese hamster brain tubulin and MAP2. We examined three anti-tubulin hybridoma antibodies (Tu3B, Tu9B, Tu12) specific for beta-tubulin, and two anti-MAP2 hybridoma antibodies (AP9,AP13). The specificity of each of the monoclonal antibodies was characterized by staining nitrocellulose electrophoretic blots of SDS-polyacrylamide gels of whole brain or hippocampal extracts. Each hybridoma antibody bound only its respective antigen in these preparations. Polyclonal antisera against tubulin were also examined. Sections reacted with antisera against tubulin or monoclonal antibodies against beta-tubulin revealed a wide variety of stained cellular compartments. The reaction product was found to decorate dendritic and axonal microtubles in neurons; glial cells were also stained. MAP2 immunoreactivity was found only in neurons. In the case of one of the monoclonal antibodies (AP9), staining was preferentially associated with dendritic processes. However, light but significant staining of axonal processes was seen with AP13. Within dendrites, MAP2 was found associated with dendritic microtubules and postsynaptic densities (psd), both in shaft and spine synapses. In addition, strong immunoreactivity for MAP2 was found within the cytoplasm of dendritic spines. There was little or no immunoreactivity for tubulin in the spine cytoplasm, although the psd was stained. The localization of MAP2 in dendritic spines and in the psd suggests that this protein may have a biological role independent of its association with microtubules. The observations on differential staining of the hybridoma antibodies against MAP2 suggest that there may be distinct subtypes or states of MAP2 within neurons.
利用针对中国仓鼠脑组织微管蛋白和微管相关蛋白2（MAP2）制备的单克隆杂交瘤抗体，通过免疫细胞化学方法分析了脑组织中微管蛋白和MAP2的分布及亚细胞定位。我们检测了三种对β-微管蛋白具有特异性的抗微管蛋白杂交瘤抗体（Tu3B、Tu9B、Tu12），以及两种抗MAP2杂交瘤抗体（AP9、AP13）。通过对全脑或海马提取物的SDS-聚丙烯酰胺凝胶的硝酸纤维素电泳印迹进行染色，对每种单克隆抗体的特异性进行了表征。在这些制剂中，每种杂交瘤抗体仅与各自的抗原结合。还检测了针对微管蛋白的多克隆抗血清。与抗微管蛋白抗血清或抗β-微管蛋白单克隆抗体反应的切片显示出多种染色的细胞区室。发现反应产物可修饰神经元中的树突和轴突微管；胶质细胞也被染色。仅在神经元中发现MAP2免疫反应性。就其中一种单克隆抗体（AP9）而言，染色优先与树突状突起相关。然而，AP13可见轴突状突起有轻微但明显的染色。在树突内，发现MAP2与树突微管和突触后致密物（psd）相关，在轴突和棘突触中均如此。此外，在树突棘的细胞质内发现了对MAP2的强免疫反应性。尽管psd被染色，但树突棘细胞质中微管蛋白的免疫反应性很少或没有。MAP2在树突棘和psd中的定位表明，这种蛋白质可能具有与其与微管的结合无关的生物学作用。针对MAP2的杂交瘤抗体的差异染色观察结果表明，神经元内可能存在不同的MAP2亚型或状态。
