人体内耳冰冻切片免疫组织化学染色技术探讨  

Study on immunohistochemical staining techniques of human inner ear frozen sections

作　　者：李颖[1,2] 陈彪[1] 王乃利[3] 郝欣平[1] Li Ying;Chen Biao;Wang Naili;Hao Xinping(Department of Otolaryngology Head and Neck Surgery,Beijing Tongren Hospital,Capital Medical University,Beijing 100730,China;Beijing Institute of Otolaryngology,Key Laboratory of Otolaryngology Head and Neck Surgery(Capital Medical University),Ministry of Education,Beijing Key Laboratory of Nasal Diseases,Beijing 100005,China;Morphological Laboratory,Teaching Center,Institute of Basic Medicine,Chinese Academy of Medical Sciences,Beijing 100005,China)

机构地区：[1]首都医科大学附属北京同仁医院耳鼻咽喉头颈外科,北京100730 [2]北京市耳鼻咽喉科研究所,耳鼻咽喉头颈科学教育部重点实验室(首都医科大学),鼻病研究北京市重点实验室,北京100005 [3]中国医学科学院基础医学研究所教学中心形态实验室,北京100005

出　　处：《首都医科大学学报》2022年第6期905-910,共6页Journal of Capital Medical University

基　　金：北京市科技计划课题(Z171100000417050)。

摘　　要：目的通过对人内耳冰冻切片免疫组织化学染色制作过程中防脱片和抗原修复方法的探讨,寻找获得满意染色效果的最佳技术方法。方法取无已知耳科疾病的尸检颞骨标本,经取材、固定、脱钙、脱水、包埋后,行冰冻切片,-20℃冷冻保存。染色前的防脱片处理分为Ⅰ组(后固定组)、Ⅱ组(烤片组)和Ⅲ组(后固定联合烤片组)。处理后,每组再行抗原修复,分别分为A组(热修复组)、B组(酶消化组)和C组(Triton组)。应用间接荧光免疫组织化学技术检测WARP、NF200、CD68及IBA1这4种抗体在各组中的表达,激光共聚焦显微镜下观察,并对其脱片情况进行结果分析。结果在防脱片处理的3组中,Ⅲ组(后固定联合烤片组)的防脱片效果最佳,基本无脱片现象。抗原修复的3组中,A组和C组的抗原修复作用较为满意,B组修复作用不佳,且对组织的细胞结构产生了破坏作用。结论应用后固定联合烤片法防止脱片和应用热修复或Triton进行抗原修复在本研究中取得了良好的效果,适当的防脱片处理和抗原修复方法对提高内耳冰冻切片免疫组织化学染色的敏感性和制片质量起着至关重要的作用。Objective To study the methods of anti-slice escaping treatment and antigenic retrieval in the process of immunohistochemical staining of frozen sections of human inner ear for an improved staining.Methods Temporal bone specimens without ear diseases were taken.After sampling,fixation,decalcification,dehydration,and embedding,frozen sections were performed and frozen at-20℃.According to the anti-slice escaping treatment,the sample was divided into three groups:post-fixation group(group I),baked slice group(groupⅡ),and post-fixation combined with baked slice group(groupⅢ).Then each group underwent antigen retrieval and was divided into group A(heat repair group),group B(enzymatic digestion group)and group C(Triton group).The expression of four antibodies of WARP,NF200,CD68 and IBA1 in each group was detected by indirect fluorescence immunohistochemistry,observed under laser confocal microscope,and the results of dissection were analyzed.Results Compared to groupⅠand groupⅡ,groupⅢhad the best effect on preventing slices escape from glass slid.The antigen retrieval effect of group A and group C was relatively satisfactory.While the repair effect of group B was not good with a destructive effect on the cellular structure of the tissue.Conclusion The application of post-fixation combined with baked slice method to anti-slice escaping and the application of heat repair or Triton treatment for antigen repair have achieved good results in this study.Appropriate anti-slice escaping treatment and antigen retrieval methods play a crucial role in improving the sensitivity of immunohistochemical staining and preparation quality of inner ear frozen sections.

关 键 词：人内耳 冰冻切片 防脱片 抗原修复 间接荧光免疫组织化学技术 

分 类 号：R764.3[医药卫生—耳鼻咽喉科]