不同厚度神经球冰冻切片免疫荧光细胞化学染色结果的比较  

Comparison of immunofluorescence cytochemical staining results in frozen sections of different-thickness neurospheres

作　　者：马雪霞 汪兆艳[2] 王倩[2] 杨印祥[2] 栾佐 MA Xuexia;WANG Zhaoyan;WANG Qian;YANG Yinxiang;LUAN Zuo(Second Clinical College,Southern Medical University,Guangzhou 510515;Department of Pediatrics,Navy General Hospital of Chinese PLA,Beijing 100048,China)

机构地区：[1]南方医科大学第二临床医学院,广东广州510515 [2]海军总医院儿科,北京100048

出　　处：《细胞与分子免疫学杂志》2018年第6期495-498,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家重点研发计划(2017YFA0104200)

摘　　要：目的探索适合免疫荧光细胞化学染色的神经球最佳冰冻切片厚度。方法选取悬浮培养10～12 d的神经球,制作厚度分别为4、7、10μm的冰冻切片。采用免疫荧光细胞化学染色比较神经球神经上皮干细胞蛋白(nestin)的表达和定位。结果培养10～12 d的神经球直径为200～250μm,状态良好,折光性强,呈圆球状,周边有毛刺。4μm的神经球冰冻切片制作较难,容易卷片,但细胞层次清楚,染色均匀,密疏适宜,可较清楚计数细胞。成像清楚。胞核清晰,可见较多nestin染色阳性的完整胞质包绕4',6-二脒基-2-苯基吲哚(DAPI)染色阳性的胞核,能观察更多的细胞间细节。7μm切片较4μm切片制作容易,切片较平整,细胞层次不如4μm切片清楚,染色较均匀。所含细胞数较4μm多,可粗略计算细胞数。荧光拍照不能完全对焦,可见部分完整细胞形态。10μm的冰冻切片制作相对容易,切片平整,但细胞层次不清楚,堆叠严重,成像不清楚,难以看到完整细胞形态。结论 4μm厚度的神经球冰冻切片较适合进行免疫荧光细胞化学染色和分析。Objective To explore the optimal frozen section thickness of neurospheres for immunofluorescence cytochemical staining. Methods We selected the neurospheres of 10-12-day suspension culture to make frozen sections of varying thickness： 4,7 and 10 μm,and then performed immunofluorescent staining to compare the expression and location of nestin. Results The diameters of the neurospheres cultured for 10-12 days were among 200-250 μm. The neurospheres were in a good condition and had a strong refractivity. The cells were spherical in shape,with burrs in the peripheral area.Practical y,it was hard to make the 4 μm frozen sections which were wrapped around themselves easily. The advantages were that,their cells had a clear-cut structure,uniform staining and practical density,thus making it easier to calculate the cell number. The imaging was clear and the nuclei were distinct,too. It could be seen that much of the whole cytoplasm,with the nestins presenting positive staining,wrapped itself around the nuclei in which the DAPI also presented positive staining,and intercellular details could be observed meanwhile. Comparatively,the 7 μm frozen sections were not that hard to get. They were quite smooth and showed rather uniform staining. The cell number turned out to be larger than that of the 4 μm frozen sections,but the cell structure was not that clear-cut,so it was difficult to make an accurate estimate of the cell number.Besides,to be in full focus could not be achieved while using fluorescent photography. Hence,only part of the entire cell morphology could be seen. The frozen slice of 10 μm is relatively easy to make,the slice is flat,but did not have a clear-cut cell structure,either. There was a serious piling phenomenon and no clear imaging. Consequently,it was hard to see the complete cell morphology. Conclusion The 4 μm frozen sections of neurospheres are more conducive to the observation and analysis of immunofluorescent staining results than those of other thicknesses.

关 键 词：神经球 冰冻切片 厚度 悬浮培养 神经上皮干细胞蛋白(nestin) 

分 类 号：Q189[生物学—神经生物学] R392-33[生物学—普通生物学]