不同固定剂对鼻息肉上皮细胞免疫荧光染色的影响

doi:10.3969/j.issn.1006-7795.2020.02.008

首都医科大学学报 ›› 2020, Vol. 41 ›› Issue (2): 189-193.doi: 10.3969/j.issn.1006-7795.2020.02.008

• 慢性鼻窦炎伴鼻息肉的基础研究 • 上一篇下一篇

不同固定剂对鼻息肉上皮细胞免疫荧光染色的影响

李颖^1,2, 矫健^1,2, 张罗^1,2

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

收稿日期:2020-01-14 出版日期:2020-04-21 发布日期:2020-04-16
通讯作者: 张罗 E-mail:dr.luozhang@139.com
基金资助:
国家自然科学基金重点项目（81630023），国家重点研发计划（2016YFC0905200），教育部长江学者及创新团队发展计划（IRT13082），北京市医院管理局使命人才计划（SML20150203），北京市自然科学基金面上项目（7172053）。

Effects of different fixatives on immunofluorescence staining in nasal polyp-derived epithelial cells

Li Ying^1,2, Jiao Jian^1,2, Zhang Luo^1,2

1. Department of Otorhinolaryngology Head and Neck Surgery, Beijing Tongren Hospital, Capital Medical University, Beijing 100730, China;
2. Beijing Institute of Otorhinolaryngology, Key Laboratory of Otorhinolaryngology Head and Neck Surgery, Ministry of Education, Beijing Key Laboratory of Nasal Diseases, Beijing 100005, China

Received:2020-01-14 Online:2020-04-21 Published:2020-04-16
Supported by:
This study was supported by National Natural Science Foundation of China （81630023）， National Key R&D Program of China （2016YFC0905200）， Program for the Changjiang Scholars and Innovative Research Team （IRT13082）， Beijing Municipal Administration of Hospitals Mission Plan （SML20150203）， Natural Science Foundation of Beijing （7172053）.

摘要/Abstract

摘要： 目的通过激光共聚焦技术，观察鼻息肉体外培养上皮细胞在3种不同固定剂作用下的免疫荧光染色效果，探讨不同固定剂对免疫荧光染色结果的影响，从而选择最佳固定方法。方法用酶消化法分离培养鼻息肉上皮细胞，接种培养7 d后，分别用4%（质量分数）多聚甲醛（paraformaldehyde，PFA）、等体积甲醇丙酮混合液及甲醇室温固定，1%（体积分数）Triton X-100透膜，山羊血清封闭，滴加一抗4 ℃过夜，滴加相应荧光二抗，含DAPI封片剂封片，激光共聚焦显微镜观察。结果等量甲醇丙酮混合液及甲醇对标记的4个标记蛋白均有较好的固定作用；4%（质量分数）PFA对跨膜蛋白16A（transmembrane protein 16A，TMEM16A）固定效果差，对紧密连接蛋白（zonular occludens-1，ZO-1）的固定效果不理想。结论甲醇丙酮混合液及甲醇对标记的4个标记蛋白的固定作用均较好，为体外培养细胞免疫荧光染色最佳固定剂的选择提供参考。

关键词: 固定剂, 上皮细胞, 免疫荧光, 激光共聚焦

Abstract: Objective To observe and compare the effects of three different fixatives on immunofluorescence staining in nasal polyp-derived epithelial cells with laser scanning confocal technique in order to choose the optimal fixation method. Methods The nasal polyp-derived epithelial cells were dissociated by enzymatic digestion method and cultured for 7 days, the cells were fixed by 4% paraformaldehyde (PFA), methanol-acetone (50:50), or methanol at room temperature, permeabilized by 1% Triton X-100, blocked with goat serum, and then incubated overnight at 4℃ with primary antibodies. Following the primary incubation, the cells were treated with the secondary antibodies labelled with fluorogenic substrates and counterstained with DAPI nuclear stain and then examined by microscopy using the confocal microscope. Results Methanol-acetone (50:50) and methanol showed a good fixation effect on four labelled proteins; however, 4% PFA showed a poor fixation effect on transmembrane protein 16A (TMEM 16A) and zonular occludens-1(ZO-1). Conclusion Methanol-acetone (50:50) and methanol showed good fixation effect on four labelled proteins and might be an ideal fixatives for cultured nasal epithelial cells.

Key words: fixative, epithelial cell, immunofluorescence, laser scanning confocal

中图分类号:

李颖, 矫健, 张罗. 不同固定剂对鼻息肉上皮细胞免疫荧光染色的影响[J]. 首都医科大学学报, 2020, 41(2): 189-193.

Li Ying, Jiao Jian, Zhang Luo. Effects of different fixatives on immunofluorescence staining in nasal polyp-derived epithelial cells[J]. Journal of Capital Medical University, 2020, 41(2): 189-193.

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不同固定剂对鼻息肉上皮细胞免疫荧光染色的影响

Effects of different fixatives on immunofluorescence staining in nasal polyp-derived epithelial cells

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