固定与透膜方法对免疫荧光检测肝脏冷冻切片中胰岛素的影响  

Effects of fixation and permeabilization protocols on immunofluores⁃cence detection of insulin in liver frozen sections

作　　者：张杰 陆仲夏 李欣宇 刘路馨 路新枝[1] ZHANG Jie;LU Zhongxia;LI Xinyu;LIU Luxin;LU Xinzhi(School of Medicine and Pharmacy,Ocean University of China,Qingdao 266003,China)

机构地区：[1]中国海洋大学医药学院,山东青岛266003

出　　处：《中国病理生理杂志》2023年第9期1716-1723,共8页Chinese Journal of Pathophysiology

基　　金：国家重点研发计划专项(No.2018YFC0311105)。

摘　　要：目的:探讨不同的固定与透膜方法对免疫荧光检测小鼠肝脏冷冻切片中胰岛素的影响,为研究胰岛素在肝脏中的代谢及胰岛素与其他分子的相互作用提供技术支持。方法:采用切片前固定或切片后固定,制作小鼠肝脏组织冷冻切片。通过间接免疫荧光技术检测5种固定与透膜方案对小鼠肝脏组织冷冻切片中胰岛素荧光染色的影响。结果:(1)方案1采用4%多聚甲醛固定联合0.5%Triton X-100透膜检测,肝血窦和肝索结构清晰,胰岛素荧光信号主要分布于细胞质膜和细胞质;(2)方案2采用4%多聚甲醛固定联合0.5%saponin透膜检测,肝血窦和肝索结构清晰,切片整体的荧光信号非常微弱,且荧光信号主要分布于细胞质膜附近;(3)方案3采用丙酮一步固定与透膜检测,肝血窦不清晰,部分肝细胞出现肿胀,胰岛素荧光信号主要分布于细胞质膜附近;(4)方案4采用甲醇一步固定与透膜检测,肝血窦不清晰,细胞间界限模糊,胰岛素荧光信号出现明显的核易位;(5)方案5采用4%多聚甲醛(含5%冰醋酸)一步固定与透膜检测,肝血窦和肝索结构清晰,胰岛素荧光信号主要分布于细胞质膜和细胞质。结论:方案1和5适于肝脏冷冻切片中胰岛素的检测。AIM:To investigate the impacts of various fixation and permeabilization methods on the immuno-fluorescence detection of insulin in frozen sections of mouse liver,as well as to offer technical assistance for investigating insulin metabolism and the interactions between insulin and other molecules in the liver.METHODS:Frozen sections of mouse liver were obtained using either pre-sectioning or post-sectioning fixation.The effects of five different fixation and permeabilization protocols on insulin fluorescence staining were examined in these liver sections using the indirect immuno-fluorescence technique.RESULTS:(1)In protocol 1,using 4%paraformaldehyde fixation paired with 0.5%Triton X-100 permeabilization resulted in well-defined structures of hepatic sinuses and hepatic cords.Insulin fluorescence signals were predominantly found in the cytoplasmic membrane and the cytoplasm.(2)In protocol 2,following 4%paraformalde-hyde fixation coupled with 0.5%saponin permeabilization,clear structures of hepatic sinuses and hepatic cords were seen.However,fluorescence signals across the sections were relatively weak and primarily localized near the cytoplasmic membrane.(3)With protocol 3,utilizing the one-step fixation and permeabilization with acetone led to unclear hepatic si-nuses and some hepatocytes exhibiting swelling.Insulin fluorescence signals were mainly concentrated near the cytoplas-mic membrane.(4)For protocol 4,using the one-step fixation and permeabilization with methanol resulted in indistinct hepatic sinuses,unclear cellular boundaries,and noticeable nuclear translocation of insulin fluorescence signals.(5)In protocol 5,employing the one-step fixation and permeabilization with 4%paraformaldehyde(inclusive of 5%acetic acid)resulted in sharply outlined structures of hepatic sinuses and hepatic cords.Insulin fluorescence signals were primarily lo-cated in the cytoplasmic membrane and the cytoplasm.CONCLUSION:Protocols 1 and 5 are deemed suitable for detect-ing insulin in liver frozen sections.

关 键 词：胰岛素 肝脏 固定 透膜 冷冻切片 免疫荧光 

分 类 号：R587.1[医药卫生—内分泌] R363[医药卫生—内科学]