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

doi:10.3969/j.issn.1006-7795.2020.02.008

Journal of Capital Medical University ›› 2020, Vol. 41 ›› Issue (2): 189-193.doi: 10.3969/j.issn.1006-7795.2020.02.008

• Basic Research on Chronic Rhinosinusitis with Nasal Polyps • Previous Articles Next Articles

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）.

Abstract

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

CLC Number:

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.

0
/ / Recommend

Add to citation manager EndNote|Reference Manager|ProCite|BibTeX|RefWorks

URL: https://journal03.magtech.org.cn/Jweb_sdykdxxb/EN/10.3969/j.issn.1006-7795.2020.02.008

https://journal03.magtech.org.cn/Jweb_sdykdxxb/EN/Y2020/V41/I2/189

References

[1] DiDonato D, Brasaemle D L. Fixation methods for the study of lipid droplets by immunofluorescence microscopy[J].J Histochem Cytochem,2003,51(6):773-780.
[2] Schmiedeberg L,Skene P,Deaton A.A temporal threshold for formaldehyde crosslinking and fixation[J].PLoS One,2009,4(2):e4636-e4638.
[3] 王伯沄,李玉松,黄高昇,等.病理学技术[M].北京:人民卫生出版社,2000:936-938.
[4] 马登磊,张旭,罗艺,等.微管相关蛋白tau转基因细胞和动物模型的建立及tau蛋白病变表征[J].首都医科大学学报,2019,40(4):582-58.
[5] Dalpé G, Leclerc N, Vallée A, et al. Dystonin is essential formaintaining neuronal cytoskeleton organization[J]. Mol Cell Neurosci,1998,10(5-6):243-257.
[6] Kast J I, Wanke K, Soyka M B,et al. The broad spectrumof interepithelial junctions in skin and lung[J]. J Allergy Clin Immunol, 2012, 130(2):544-547.
[7] Suzuki T. Regulation of intestinal epithelial permeability by tight junctions[J]. Cell Mol Life Sci,2013,70(4):631-659.
[8] Jesaitis L A,Goodenough D A. Molecular characterization and tissue distribution of ZO-|2, a tight junction protein homologous to ZO-1 and the Drosophila discs-large tumor suppressor protein[J].J Cell Biol,1994,124(6):949-961.
[9] Caputo A,Caci E,Ferrera L,et al. TMEM16A,a membrane protein associated with calcium-dependent chloride channel activity[J]. Science, 2008,322(5901):590-594.
[10] Huang F,Zhang H,Wu M,et al. Calcium-activated chloride channel TMEM16A modulates mucin secretion and airway smooth muscle contraction[J]. Proc Natl Acad Sci U S A,2012,109(40):16354-16359.
[11] Catalan M A,Kondo Y,Peña-Munzenmayer G, et al. A fluid secretion pathway unmasked by acinar-specific Tmem16A gene ablation in the adult mouse salivary gland[J]. Proc Natl Acad Sci U S A,2015,112(7):2263-2268.
[12] Crutzen R,Virreira M,Markadieu N, et al. Anoctamin 1(Ano1) is required for glucose-induced membrane potential oscillations and insulin secretion by murine β-cells[J]. Pflugers Arch,2016,468(4):573-591.
[13] Schreiber R,Faria D, Skryabin B V, et al. Anoctamins support calcium-dependent chloride secretion by facilitating calcium signaling in adult mouse intestine[J]. Pflugers Arch, 2015, 467(6):1203-1213.
[14] Kawasaki H, Altieri D C, Lu C D, et al. Inhibition of apoptosis by survivin predicts shorter survival rates in colorectal cancer[J]. Cancer Res, 1998, 58(22):5071-5074.
[15] Ler S Y, Leung C H, Khin L W, et al.HDAC1 and HDAC2 independently predict mortality in hepatocellular carcinoma by a competing risk regression model in a Southeast Asian population[J].Oncol Rep, 2015, 34(5):2238-2250.

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

PDF

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	Li Ying, Chen Biao, Wang Naili, Hao Xinping. Study on immunohistochemical staining techniques of human inner ear frozen sections [J]. Journal of Capital Medical University, 2022, 43(6): 905-910.
[2]	Chen Xiaomei, Ma Yuanyuan, Nie Jing, Zhu Fengxin. Progress on the injury and repair mechanism of renal tubular epithelial cells in the transition of acute kidney injury to chronic kidney disease [J]. Journal of Capital Medical University, 2022, 43(5): 720-727.
[3]	Duan Nan, Liu Yi, Li Haixia[KG-*2], Jiao Lili. Effects of glycated albumin on inducing injury and inflammation in human proximal tubular epithelial cells [J]. Journal of Capital Medical University, 2017, 38(2): 289-294.
[4]	Li Jinghua, Wang Ming, Zhang Rui, Feng Limin. Effect of the miR-886-5p on the cervical squamous epithelial cell clone formation and cervical cancer chemotherapy [J]. Journal of Capital Medical University, 2015, 36(6): 929-935.
[5]	Lyu Xiaohong, Wang Lei, Liu Chang, Wang Yingying, Yang Jinkui. Effects and possible mechanisms of pancreatic duct epithelial cells on insulin secretion of the islet β cells [J]. Journal of Capital Medical University, 2014, 35(1): 77-80.
[6]	YAN Shao-fei;WEN Zhao-yang;GAO Er-jing;NIU Jing;ZHENG Shao-peng;DING Wei. Late endosome sorting contributed to the transduction activation of type 2 adeno-associated virus in IB3 transformed human bronchial epithelial cells [J]. Journal of Capital Medical University, 2011, 32(4): 519-524.
[7]	DOU Zhang-feng;ZHANG Yue;JI Tuo;XUE Hong;ZHU Jin-xia. Differential Expression of NKCC1 and NKCC2 in Rat Gastric Mucosa [J]. Journal of Capital Medical University, 2011, 32(2): 214-217.
[8]	Shi Linggai;Han Demin;Zhang Luo;. Effect of ATP on Human Nasal Ciliary Beat Frequency [J]. Journal of Capital Medical University, 2009, 30(1): 57-61.
[9]	Bo Yuhong;Jiang Feng;Bi Zengqi. Clinical Relevance of Urinary Epithelial Cells Adhesive with Bacteria for Diagnosis of UTI in Old Women [J]. Journal of Capital Medical University, 2009, 30(1): 97-99.
[10]	Liu Dongshan;Zhang Guizhi;Jin Yinlong. Volatile Organic Compounds(VOCs)-Induced Acute Cytotoxicity to Human Bronchial Epithelial Cell Line BEAS-2B [J]. Journal of Capital Medical University, 2005, 26(6): 662-666.
[11]	Li Juehua;Wan Hong;Zhai Jing;Wang Zhongcheng. Free-Serum Culture Method to Remove Fibroblast in Nerve Tissue [J]. Journal of Capital Medical University, 2004, 25(2): 205-207.
[12]	Li Junhua;Wan Hong;Wang Zhongcheng Beijing Neurosurgical Institute. Tubulin-β Expression of Various Culture Cells Using Immunoinfluorescence [J]. Journal of Capital Medical University, 2004, 25(1): 57-59.
[13]	Yang Yaping;Zhu Xinping;Yang Jing;Lei Liping;Jing Peng. Immunohistochemical Localization of Ts87 Antigen of Trichinella Spiralis [J]. Journal of Capital Medical University, 2003, 24(2): 108-110.
[14]	Liu Zhen;Li Guojun;Zhang Jie;Zhou Chunyi;Li Penggao;Zhao Chunli;Yang Hui;Wei Zheng;Jing Peng. Effects of Manganese with Different Valences on Mitochondrial Membrane Potential in Human SH-SY5Y Cells in Vitro [J]. Journal of Capital Medical University, 2002, 23(1): 14-16.
[15]	Hong Mingli;Huang Xianghong;Li Yunsheng;Fu Zhifeng;Wang Zhengming;Ren Jiaqiang;Tong Jie;Chen Daili;Qin Bin;Zhang Tong;Li Fei;Su Jianxin. Pathologic Study on Pathogenesis of Experimental Biliary Cirrhosis [J]. Journal of Capital Medical University, 1996, 17(1): 36-39.