大鼠延髓腹外侧A1/C1区儿茶酚胺能神经元的电镜观察——包埋前液氮冻融和Triton X-100组织处理方法的比较  

Electron microscopic observation of A1/C1 catecholamine neurons immunoreacted in the rostroventrolateral medulla of rats——A comparative study of pre-embedding treatments of tissues with liquid nitrogen freeze-thaw or Triton X-100

作　　者：梁卫华[1,2] 亢君君[3] 黄晓峰[1] 刘莹莹[3] 

机构地区：[1]第四军医大学病理学与病理生理学教研室 [2]68057部队 [3]第四军医大学神经生物学教研室

出　　处：《神经解剖学杂志》2015年第6期799-803,共5页Chinese Journal of Neuroanatomy

基　　金：国家自然科学基金(31171102;31471097)

摘　　要：目的:比较液氮冻融和Triton X-100不同方法对大鼠延髓腹外侧A1/C1区儿茶酚胺能神经元多巴胺β羟化酶(dopamine-β-hydroxylase,DβH)免疫反应(immunoreaetive,ir)标记的影响。方法:实验分组包括:液氮冻融1次组、液氮冻融2次组、0.03%Triton X-100组、0.05%Triton X-100组。应用包埋前纳米金-银加强免疫电镜技术,标记延髓腹外侧A1/C1区DβH-ir神经元。结果:光镜下可观察到DβH-ir产物分布于胞体和突起,清晰标记A1/C1区神经元。不同组间DβH-ir神经元数量明显不同:冻融组数量少,突起少且细短;Triton X-100组数量明显增多,突起浓密且长,其中以0.05%Triton X-100组最为明显,差异显著。电镜结果显示:DβH-ir金-银颗粒主要分布在神经元胞体、树突和轴突终末内,DβH-ir轴突终末与胞体或树突形成密切接触或非对称性突触。金颗粒标记的数量在Triton X-100组明显多于冻融组这与光镜观察结果相一致。结论:包埋前免疫电镜技术中应用少量Triton X-100处理,可取代液氮冻融,能更好地增加胞膜的通透性,提高抗体的组织渗透能力和特异性标记是一种较好的包埋前免疫电镜的处理方法。Objective：The present study was aimed to explore effects between different pre-embedding treatments of liquid nitrogen freeze-thaw and Triton X-100 via detection of dopamine-β-hydroxylase（DβH） immunoreactivity in Al/Cl catecholamine neurons in the ventrolateral medulla oblongata of rats.Methods：Brainstem sections were grouped as liquid nitrogen freeze-thaw for once or twice,and 0.03%or 0.05%TritonX-100,respectively.A pre-embedding nano-gold/silver enhancement immunoelectron microscopic technique was applied to detect DβH immunoreactive（ir） product in Al /C1 area.Results：DβH-ir product was distributed in somas and processes,clearly outlining Al/Cl neurons.DβH-ir neurons were distinctly different in the number and the shape in freeze-thaw groups as compared to Triton X-100 groups.A significant increase in DβH-ir neurons was evident in Triton X-100 groups,especially in 0.05%group that neurons born dense and long processes,whereas short and thin in freeze-thaw groups.Gold particles indicative of DβH immunoreactivity were localized to somas,axonal terminals in Al/Cl neurons under the electronmicroscope.DβH-ir terminals were found in contacts with somas and dendrites.Asymmetric synaptic contacts were visualized between them.Gold particles were much denser in DβH-ir neurons in Triton X-100 groups than those in freeze-thaw groups,being in consistence with the light microscope observation.Conclusion：Pre-embedding Triton X-100 treatment in immunoelectron microscopic labeling may replace liquid nitrogen freeze-thaw treatment to enhance membrane permeability,leading to better antibody penetration and specific antigen combination.

关 键 词：多巴胺Β羟化酶 免疫电镜技术 液氮冻融 Triton X-100 大鼠 

分 类 号：R338[医药卫生—人体生理学]