Steen P, Kalghatgi L, Constantine-Paton M
Department of Biology, Yale University, New Haven, Connecticut 06511.
J Comp Neurol. 1989 Nov 15;289(3):467-80. doi: 10.1002/cne.902890311.
Few immunocytochemical probes have been developed for cold-blooded vertebrates, thus hampering analyses of cellular processes in these species. Those developed from mammalian and avian tissue often fail either to react or to show similar specificities in poikilotherms. Therefore, we have begun raising monoclonal antibodies (mabs) in mice against frog and tadpole brain tissue. The following analyses of two of these mabs suggest that these antibodies represent specific probes for frog axons and oligodendrocytes. Mab Olig recognizes all the myelinated axon tracts of the mature frog brain and spinal cord, as well as the tracts of the developing tadpole CNS once they have become myelinated. Axons cut in cross section show characteristic o-shaped staining around individual axons when processed with this antibody. Particularly easy to visualize in the tadpole are immunoreactive cell bodies and processes, seen in continuity with the myelin sheath. Occasionally, in this developing tissue, cells with highly branched processes characteristic of immature oligodendrocytes are observed. No other cells or processes within the brain or spinal cord react with this antibody. Mab Linc stains numerous filaments in all axonal projections. Occasionally, a thin rim of filamentous staining is observed in cell somata, but many regions rich in neuronal somata or dendrites are unreactive to this antibody. This in vivo staining pattern suggests that the Linc antigen is differentially distributed within neurons and exhibits a high concentration in axons. Linc immunoreactivity is robust in the processes of a subpopulation of dissociated tectal cells in culture. These Linc-positive cells are characterized as neurons on morphological criteria. Also, intense Linc immunoreactivity is present in the bundles of retinal axons that extend from retinal explants. Olig immunoreactivity, however, has not been detected in tectal cultures or retinal explants. Improved staining following Triton X-100 treatment of tissue sections suggests that neither of the mabs recognizes lipid antigens and that both are probably localized within the cell cytoplasm. Only the Linc mab reacts on Western blots of denatured brain protein. Linc consistently recognizes two Triton X-100-insoluble proteins with apparent molecular weights of 56 and 58 kD. The epitopes recognized by the Olig and Linc mabs have been surveyed in terms of their resistance to optic nerve crush and their consequent value in studies requiring such procedures. Possible homologies to known cell-type-specific molecules are discussed.
针对冷血脊椎动物开发的免疫细胞化学探针很少，这阻碍了对这些物种细胞过程的分析。那些从哺乳动物和鸟类组织开发的探针在变温动物中往往要么不发生反应，要么显示出不同的特异性。因此，我们开始在小鼠中制备针对青蛙和蝌蚪脑组织的单克隆抗体（mab）。对其中两种单克隆抗体的以下分析表明，这些抗体代表了针对青蛙轴突和少突胶质细胞的特异性探针。单克隆抗体Olig识别成熟青蛙脑和脊髓的所有有髓轴突束，以及发育中的蝌蚪中枢神经系统一旦形成髓鞘后的束。用该抗体处理时，横切的轴突在单个轴突周围显示出特征性的o形染色。在蝌蚪中特别容易观察到免疫反应性细胞体和突起，它们与髓鞘连续。偶尔，在这个发育中的组织中，会观察到具有未成熟少突胶质细胞特征的高度分支突起的细胞。脑或脊髓内没有其他细胞或突起与该抗体发生反应。单克隆抗体Linc在所有轴突投射中染色许多细丝。偶尔，在细胞体中观察到细丝状染色的薄边缘，但许多富含神经元细胞体或树突的区域对该抗体无反应。这种体内染色模式表明，Linc抗原在神经元内分布不同，在轴突中浓度较高。在培养的解离视顶盖细胞亚群的突起中，Linc免疫反应性很强。根据形态学标准，这些Linc阳性细胞被鉴定为神经元。此外，从视网膜外植体延伸出的视网膜轴突束中存在强烈的Linc免疫反应性。然而，在视顶盖培养物或视网膜外植体中未检测到Olig免疫反应性。用Triton X-100处理组织切片后染色的改善表明，这两种单克隆抗体都不识别脂质抗原，并且两者可能都定位于细胞质内。只有Linc单克隆抗体在变性脑蛋白的免疫印迹上发生反应。Linc始终识别两种Triton X-100不溶性蛋白，其表观分子量分别为56和58 kD。已根据它们对视神经挤压的抗性以及它们在需要此类操作的研究中的价值，对Olig和Linc单克隆抗体识别的表位进行了研究。讨论了与已知细胞类型特异性分子的可能同源性。
