大鼠脑组织冰冻切片BrdU/DCX免疫荧光双染优化条件探讨  

Optimized condition exploration of BrdU/DCX double immunofluorescence staining on frozen sections of rat brain tissue

作　　者：高家乐 姚明江[1] 张伟[2] 杨斌[2] 刘建勋[1] Gao Jiale;Yao Mingjiang;Zhang Wei;Yang Bin;Liu Jianxun(Beijing Key Laboratory of Pharmacology of Chinese Materia Medica,Institute of Basic Medical Sciences of Xiyuan Hospital,China Academy of Chinese Medical Sciences,Bejing 100091,China;Department of Pathology,Xiyuan Hospital,China Academy of Chinese Medical Sciences,Bejing 100091,China)

机构地区：[1]中国中医科学院西苑医院基础医学研究所,中药药理北京市重点实验室,北京100091 [2]中国中医科学院西苑医院病理科,北京100091

出　　处：《神经解剖学杂志》2022年第4期440-446,共7页Chinese Journal of Neuroanatomy

基　　金：国家自然科学基金(82030124);中国中医科学院科技创新工程“脑病学”学科重大攻关项目(C12021A01305);国家中医药管理局中医药创新团队及人才支持计划项目(ZYYCXTD-C-202007)。

摘　　要：目的:探讨在脑组织冰冻切片上进行溴脱氧尿嘧啶核苷(BrdU)和双皮质素(DCX)免疫荧光双染的方法,并在此基础上进一步优化。方法:选取雄性SD大鼠脑组织的冰冻切片,通过漂片法,利用大鼠抗BrdU抗体和兔抗DCX抗体两种不同种属来源的一抗在混合孵育和分开孵育的条件下分别进行免疫荧光双染,采集图像并观察实验结果。结果:混合孵育法BrdU阳性标记定位准确,但与之双标的DCX未能正常显色;先孵育抗BrdU抗体后孵育抗DCX抗体:BrdU阳性标记定位准确,但与之双标的DCX仍未能正常显色;先孵育抗DCX抗体后孵育抗BrdU抗体:BrdU和DCX均可正常显色,DCX荧光亮度偏暗,在此基础上提高抗DCX抗体的比例,荧光亮度适中,效果较理想。结论:结果表明,抗BrdU抗体和抗DCX抗体混合孵育后,DCX未能正常显色,可能与两种一抗产生拮抗有关,先孵育抗DCX抗体后孵育抗BrdU抗体是BrdU/DCX荧光双染一种较理想的方法。Objective:This study investigated the BrdU/DCX double immunofluorescence staining on frozen sections of brain tissue and was further optimized on this basis.Methods:During the experiment,frozen sections of male SD rat brain tissue were selected.Through the bleach section method,double immunofluorescence staining was performed respectively using two different genus sources of primary antibodies including rat anti-BrdU antibody and rabbit anti-DCX antibody under the conditions of mixed and separate incubation.After that,the images were collected and the experimental results were observed.Results:Mixed incubation:BrdU positive marker was positioned accurately,but the dual-target DCX failed to show the color normally;Incubation of anti-BrdU antibody prior to the anti-DCX antibody:BrdU positive labeling was positioned accurately,but the dual-target DCX failed again;Incubation of anti-DCX antibody prior to the anti-BrdU antibody:Both of them could show the color normally with slightly dark fluorescence brightness of DCX.With the increasing proportion of anti-DCX antibody,the fluorescence brightness is moderate,and the effect is ideal.Conclusion:The results showed that the failure of DCX after the mixed incubation of anti-BrdU antibody and anti-DCX antibody may be related to the antagonism between primary antibodies.And the incubation of anti-DCX antibody prior to the anti-BrdU antibody is a relatively ideal method for BrdU/DCX double immunofluorescence staining.

关 键 词：溴脱氧尿嘧啶核苷 双皮质素 免疫荧光双染 漂片法 脑组织 冰冻切片 大鼠 

分 类 号：O657.3[理学—分析化学] R361.2[理学—化学]