摘要
目的选择三种较常见的组织透明化方法,即PACT、MACS和Visikol,比较其在脑组织不同抗原类型和不同标记方案中的兼容性,为检测特定靶蛋白的最适组织透明化方案提供参考。方法分别利用转基因小鼠来源的内源性荧光、小分子荧光标记物标记特定蛋白、免疫荧光化学标记特定靶抗原等方法进行脑组织样本的标记;采用基于水凝胶包埋的PACT方法、基于尿素透明化技术改进的MACS方法和基于溶剂透明技术的Visikol HISTO商品化试剂三种方案对上述标记的脑组织样本分别进行透明化处理;利用共聚焦显微镜,选择适配于以上三种透明化方案样本折射率的物镜进行成像。结果以厚脑片(150μm)为研究对象,三种方法达到组织透明效果所需的时间PACT法最长,Visikol法次之,MACS法最短。对于不同内源性荧光蛋白,PACT和MACS法能较好保存抗原荧光,而Visikol法处理后荧光淬灭严重;对于以血管凝集素为例的小分子荧光标记物,三种方法均可完成标记,但Visikol法效果最好。对于胞核与胞质抗原分别进行免疫荧光化学染色,三种方法均可完成标记;而对于胞膜抗原进行免疫荧光化学染色,PACT和MACS法能较好实现标记。结论由于抗原类型的特点和透明化原理的不同,组织成像效果并不完全相同。选择合适的抗原标记方案结合特定的组织透明化方法对于成功实现一定空间范围内的三维组织成像至关重要。
Objective To compare the compatibility of three optical tissue clearing techniques,PACT,MACS and Visikol,in different antigen types and different labeling schemes in brain tissues,so as to provide reference for the optimal optical tissue clearing scheme for the detection of specific target proteins.Methods Brain tissue samples were labeled by endogenous fluorescence from transgenic mice,small-molecule fluorescent markers for specific proteins,and immunofluorescence chemical markers for specific target antigens.PACT method based on hydrogel embedding,MACS method based on urea tissue clearing technique and Visikol HISTO commercial reagents based on solvent tissue clearing technique were used in the labeled brain tissue samples for tissue clearing.The confocal microscope was used to select the objective lens suitable for the sample refractive index of the above three tissue clearing schemes for imaging.Results The duration of tissue clearing for thick brain slices(150μm)was longest in PACT and shortest in MACS.For different endogenous fluorescent proteins,PACT and MACS methods could preserve the antigen fluorescence well,while Visikol method leaded to serious fluorescence quenching.For small-molecule fluorescent markers,taking lectin as an example,all three methods could complete labeling,but Visikol method had the best effect.Immunofluorescence staining was performed for nuclear and cytoplasmic antigens,and all three methods could complete labeling.PACT and MACS methods could be used for immunofluorescence staining of cell membrane antigens.Conclusion Due to the characteristics of antigen types and the principle of different tissue clearing techniques,the results of 3D imaging for thick brain slices under different tissue clearing methods are not exactly the same.It is very important to select appropriate antigen labeling scheme combined with specific tissue clearing method for successful 3D tissue imaging in a certain spatial range.
作者
黄义源
杨亮
袁洁
智娜
刘育明
张明
赵湘辉
HUANG Yiyuan;YANG Liang;YUAN Jie;ZHI Na;LIU Yuming;ZHANG Ming;ZHAO Xianghui(School of Life Sciences&Research Center for Resource Peptide Drugs,Shaanxi Engineering&Technological Research Center for Conversation&Utilization of Regional Biological Resources,Yan an University,Yan an 716099,China;College of Life Sciences,Northwest University,Xi'an 710127,China;Department of Neurobiology,School of Basic Medical Sciences,Air Force Medical University,Xi'an 710032,China)
出处
《空军军医大学学报》
CAS
2022年第8期931-938,共8页
Journal of Air Force Medical University
基金
国家自然科学基金面上项目(82071271)
科技部科技创新2030-“脑科学与类脑研究”重大项目(2021ZD0201700)。
关键词
组织透明技术
脑
免疫荧光化学染色
三维成像
optical tissue clearing techniques
brain
immunofluorescence staining
three-dimensional imaging
