豚鼠耳蜗Corti器铺片方法改进与免疫荧光染色比较研究  

Modified Organ of Corti Whole-Mount and Immunofluorescence Staining in Guinea Pigs

作　　者：方舒 胡一勇 吕萍[1] 于宁[2] FANG Shu;HU Yiyong;LV Ping;YU Ning(Department of Otorhinolaryngology,North Sichuan Medical College Affiliated Hospital,Nanchong,637000,China;Department of Otolaryngology-Head and Neck Surgery,Institute of Otolaryngology,Chinese PLA General Hospital,Beijing 100853,China)

机构地区：[1]川北医学院附属医院耳鼻咽喉科,南充637000 [2]解放军总医院耳鼻咽喉头颈外科耳鼻咽喉研究所,北京100853

出　　处：《中华耳科学杂志》2018年第4期499-502,共4页Chinese Journal of Otology

基　　金：国家973重大科学研究计划项目(2014CB943002);国家自然科学基金项目(81470700);四川省教育厅课题(16ZB0231)~~

摘　　要：目的提高本实验室豚鼠耳蜗基底膜免疫荧光染色铺片的实验效率,为内耳研究探索一种更为快捷的免疫荧光染色方法。方法 15只成年Dunkin Hartley豚鼠注射过量1%戊巴比妥钠处死,断头取双侧耳蜗,右侧为对照组,采用常规耳蜗基底膜免疫荧光染色铺片方法,左侧为实验组,采用减少了组织固定、免疫荧光染色一抗孵育及漂洗时间的改良耳蜗基底膜免疫荧光染色铺片方法,激光共聚焦扫描显微镜在20倍、40倍、63倍镜下观察并比较两组经荧光素标记的基底膜Corti器上的肌球蛋白7a或神经丝蛋白的表达情况。结果对照组耗时37.5小时,实验组耗时5.25小时。两种染色方法均能清楚显示肌球蛋白7a或神经丝蛋白的表达情况,成像效果未见明显差异。结论快速耳蜗基底膜铺片免疫荧光染色方法耗时短、可重复性好,值得在耳科学基础研究尤其是内耳形态研究领域推广。Objective To report an improved immunofluorescence staining technique for the purpose of improving efficiency of cochlear basilar membrane whole-mount preparation and immunofluorescence staining in guinea pigs.Methods Fifteen healthy adult Dunkin Hartley guinea pigs were sacrificed by injecting overdose pentobarbitone sodium and decapitated,and the cochlea removed for immunofluorescence staining.The right cochlea(control)was processed with the routine method,while the left cochlea was processed according to a modified cochlear basilar membrane whole-mount and immunofluorescence protocol in which the time of fixation,primary antibody incubation and rinsing was reduced.The expression and location of myosinⅦa or neurofilament in the Corti’s organ were examined and compared under a confocal laser scanning microscope using a 20x,40x,or 63x glycerin-immersion lens.Results It took 37.5 h to complete all procedural steps in the routine protocol while the modified protocol required only 5.25 h.The confocal photomicrographs obtained using the two protocols both showed clear expression and location of myosin VIIa or neurofilament,with no difference in image quality.Conclusion The results indicate that the modified technique,with its time-efficiency and excellent reproducibility,is worth for adoption in inner ear research,especially in for morphological studies.

关 键 词：免疫荧光染色 耳蜗 CORTI器 豚鼠 

分 类 号：R764[医药卫生—耳鼻咽喉科]