Ito T, Nozawa A, Usuda Y, Kitamura H, Kanisawa M
Department of Pathology, Yokohama City University School Medicine, Japan.
Histochem Cell Biol. 1995 Nov;104(5):357-62. doi: 10.1007/BF01458129.
Pulmonary endocrine cells of Syrian golden hamster were stained for neural cell adhesion molecule (NCAM) with indirect fluorescent immunostaining and observed with a confocal laser scanning microscope equipped with an argon laser. Sections 100 microns thick of hamster lung fixed with phosphate-buffered 4% paraformaldehyde were prepared. The sections were incubated with rat monoclonal antibody against NCAM, followed by fluorescence-labeled antibody against rat immunoglobulin. Some were doubly immunostained for NCAM and one of the following endocrine markers: neuron-specific enolase, calcitonin gene-related peptide and serotonin. Expression of NCAM in the hamster airway epithelium was seen in cell nests resembling neuroepithelial bodies (NEBs). NCAM immunostaining was positive at the lateral cell borders between the cells composing the nest, but negative at the border with the adjacent, presumably non-endocrine cells. Double immunostaining confirmed that the grouped cells with NCAM immunoreactivity were of an endocrine nature, but that single endocrine cells did not show NCAM immunoreactivity. An electron microscopic study with NCAM immunostaining confirmed the light microscopic study. These suggest that NCAM expression could be important for the morphogenesis of NEBs. A confocal laser microscope was used to make three-dimensional images of NEBs after NCAM immunostaining and the spatial interaction between NEBs and the surrounding microenvironment was studied.
采用间接荧光免疫染色法对叙利亚金黄地鼠的肺内分泌细胞进行神经细胞黏附分子(NCAM)染色,并用配备氩离子激光的共聚焦激光扫描显微镜进行观察。制备用4%多聚甲醛磷酸盐缓冲液固定的100微米厚的地鼠肺切片。切片先用抗NCAM的大鼠单克隆抗体孵育,然后用抗大鼠免疫球蛋白的荧光标记抗体孵育。部分切片对NCAM与以下内分泌标志物之一进行双重免疫染色:神经元特异性烯醇化酶、降钙素基因相关肽和5-羟色胺。在类似神经上皮小体(NEBs)的细胞巢中可见地鼠气道上皮中NCAM的表达。NCAM免疫染色在构成巢的细胞之间的细胞侧边界处呈阳性,但在与相邻的、可能是非内分泌细胞的边界处呈阴性。双重免疫染色证实,具有NCAM免疫反应性的成组细胞具有内分泌性质,但单个内分泌细胞不显示NCAM免疫反应性。对NCAM免疫染色进行的电子显微镜研究证实了光学显微镜研究的结果。这些表明NCAM表达可能对NEBs的形态发生很重要。在NCAM免疫染色后,用共聚焦激光显微镜制作NEBs的三维图像,并研究NEBs与周围微环境之间的空间相互作用。