Schnitzer J, Kim S U, Schachner M
Brain Res. 1984 Jul;317(1):21-32. doi: 10.1016/0165-3806(84)90136-6.
Monoclonal antibody N1 reacts by indirect immunofluorescence with the cell surface of tetanus toxin-positive neurons from early postnatal mouse cerebellum. In freshly trypsinized single cell suspensions from early postnatal mouse cerebellum, 5-10% of all viable cells express N1 antigen on their surface. After 3-24 h of maintenance in vitro all N1 antigen-positive cells are tetanus toxin-positive. After culture periods of 3-4 days, most (approximately 90%) tetanus toxin-positive cells express N1 antigen on their surface. When horse serum-supplemented medium (HSSM) is used for cultivation, neurons begin to lose N1 antigen from their surface after about one week in vitro, until after two weeks in vitro, N1 antigen is no longer detectable, although some tetanus toxin-positive neurons can be shown to survive in culture. In defined medium, however, N1 antigen-positive neurons can still be detected after 34 days in vitro, the longest culture period examined so far. Complement-dependent immunocytolysis deletes all N1 antigen-positive and approximately 90% of all tetanus toxin-positive neurons from cultures. The remaining neurons reveal a morphology different from the one of the majority of small neurons, the granule cells. They have slightly larger cell bodies and several branched and unbranched cellular processes. Neonatal cerebellar cells show the same temporal sequence of appearance and disappearance of N1 antigen on most tetanus toxin-positive neurons in HSSM, and a persistence of N1 antigen on neurons in defined medium. N1 antigen becomes first detectable at embryonic day 17, and never becomes detectable in cell cultures derived from cerebella of younger mice. At all stages studied, N1 antigen expression is restricted to tetanus toxin-positive neurons, while it is absent from the cell surfaces of astrocytes, oligodendrocytes and fibroblasts. N1 antigen is also found in cultures derived from early postnatal mouse cerebrum, but is not detected in cultures derived from mouse retina, spinal cord, dorsal root ganglion, and embryonic telencephalon. It is also not detectable in cerebellar cultures from rabbit, rat, chicken and human. When N1 antibody is applied to fixed cultures where intracellular antigens are accessible, all cell types are labeled intracellularly, with astrocytes and fibroblasts revealing a fibrillary, vimentin-like staining pattern.
单克隆抗体N1通过间接免疫荧光法与出生后早期小鼠小脑破伤风毒素阳性神经元的细胞表面发生反应。在出生后早期小鼠小脑新鲜胰蛋白酶消化的单细胞悬液中，所有活细胞中有5%-10%的细胞表面表达N1抗原。在体外培养3-24小时后，所有N1抗原阳性细胞均为破伤风毒素阳性。培养3-4天后，大多数（约90%）破伤风毒素阳性细胞表面表达N1抗原。当使用补充马血清的培养基（HSSM）进行培养时，神经元在体外培养约一周后开始从其表面丢失N1抗原，直到体外培养两周后，虽然一些破伤风毒素阳性神经元在培养中仍可存活，但已无法检测到N1抗原。然而，在限定培养基中，体外培养34天后（这是迄今为止检测的最长培养时间）仍可检测到N1抗原阳性神经元。补体依赖性免疫细胞溶解从培养物中清除所有N1抗原阳性细胞以及约90%的所有破伤风毒素阳性神经元。其余神经元呈现出与大多数小神经元即颗粒细胞不同的形态。它们的细胞体稍大，有几个分支和未分支的细胞突起。新生小鼠小脑细胞在HSSM中大多数破伤风毒素阳性神经元上N1抗原出现和消失的时间顺序相同，并且在限定培养基中神经元上N1抗原持续存在。N1抗原在胚胎第17天首次可检测到，在来自更年幼小鼠小脑的细胞培养物中从未可检测到。在所有研究阶段，N1抗原表达仅限于破伤风毒素阳性神经元，而在星形胶质细胞、少突胶质细胞和成纤维细胞的细胞表面不存在。N1抗原也存在于出生后早期小鼠大脑来源的培养物中，但在来自小鼠视网膜、脊髓、背根神经节和胚胎端脑的培养物中未检测到。在来自兔、大鼠、鸡和人的小脑培养物中也无法检测到。当将N1抗体应用于可接触细胞内抗原的固定培养物时，所有细胞类型在细胞内都被标记，星形胶质细胞和成纤维细胞呈现出纤维状、波形蛋白样的染色模式。
