不同固定液对体外细胞系荧光蛋白淬灭及对胞内蛋白荧光染色的影响
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摘要
目的:比较不同细胞固定液对荧光蛋白淬灭情况以及核内、胞浆蛋白免疫荧光染色的影响。方法:分别对融合了RFP和GFP基因的鼻咽癌HK1细胞采用95%乙醇、75%乙醇、甲醇、丙酮:甲醇=1:1、5%冰乙酸、Carnoy固定液进行固定,然后采用免疫荧光法对细胞进行免疫染色。结果:六种固定液均能使荧光蛋白猝灭。免疫荧光染色方面,对于核蛋白染色,75%乙醇、95%乙醇、丙酮:甲醇=1:1、Carnoy固定液固定后核区获得明显的荧光染色,而采用甲醇、5%冰乙酸固定后荧光染色不明显。对于胞质蛋白染色,按荧光染色的清晰程度分为固定于Carnoy固定液>丙酮:甲醇=1:1>甲醇>5%冰乙酸>75%乙醇>95%乙醇,前四者固定可见分布于胞质,75%乙醇或95%乙醇固定的目标蛋白定位不清。结论:六种不同的固定液在有效失活荧光蛋白的情况下对核蛋白及胞浆蛋白抗原性的影响略有不同,可根据研究目的蛋白表达的部位及特点来选用合适的固定液。
Objective: Comparing the effects of different cell fixatives on the quenching of fluorescent proteins and the nuclear and cytoplasmic protein antigenicity. Methods: Nasopharyngeal carcinoma HK1 cells fused with RFP and GFP genes were fixed with95% ethanol, 75% ethanol, methanol, acetone: methanol=1:1, 5% glacial acetic acid, Carnoy'fixative, respectively. All cells were stained by immunofluorescence method. Results: Six fixation buffers can quench the fluorescent protein. For immunofluorescence staining,for nucleoprotein staining, 75 % ethanol, 95 % ethanol acetone: methanol = 1:1 fixation, Carnoy's fixative fixed nuclear staining after obtaining significant fluorescent staining. The fluorescence signal was weakened by fixation with methanol, 5 % glacial acetic acid. For cytoplasmic protein staining, the performed according to the clarity of fluorescent staining was Carnoy's fixative > acetone: methanol = 1:1 > methanol fixation > 5 % glacial acetic acid > 75 % ethanol > 95 % ethanol, the first four were fixedly distributed in the cytoplasm, 75 %ethanol and 95 % ethanol was fixedly distributed in the nucleus. Conclusions: Six different fixatives have slightly different effects on the antigenicity of nuclear protein and cytosolic protein in the case of effective inactivation of fluorescent protein. The appropriate fixative can be selected according to the location and characteristics of the protein expression of the research object.
Objective: Comparing the effects of different cell fixatives on the quenching of fluorescent proteins and the nuclear and cytoplasmic protein antigenicity. Methods: Nasopharyngeal carcinoma HK1 cells fused with RFP and GFP genes were fixed with95% ethanol, 75% ethanol, methanol, acetone: methanol=1:1, 5% glacial acetic acid, Carnoy'fixative, respectively. All cells were stained by immunofluorescence method. Results: Six fixation buffers can quench the fluorescent protein. For immunofluorescence staining,for nucleoprotein staining, 75 % ethanol, 95 % ethanol acetone: methanol = 1:1 fixation, Carnoy's fixative fixed nuclear staining after obtaining significant fluorescent staining. The fluorescence signal was weakened by fixation with methanol, 5 % glacial acetic acid. For cytoplasmic protein staining, the performed according to the clarity of fluorescent staining was Carnoy's fixative > acetone: methanol = 1:1 > methanol fixation > 5 % glacial acetic acid > 75 % ethanol > 95 % ethanol, the first four were fixedly distributed in the cytoplasm, 75 %ethanol and 95 % ethanol was fixedly distributed in the nucleus. Conclusions: Six different fixatives have slightly different effects on the antigenicity of nuclear protein and cytosolic protein in the case of effective inactivation of fluorescent protein. The appropriate fixative can be selected according to the location and characteristics of the protein expression of the research object.
引文
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[3]Cui Y,Gao C,Zhao Q,et al.Using Fluorescent Protein Fusions to Study Protein Subcellular Localization and Dynamics in Plant Cells[J].Methods Mol Biol,2016,1474:113-123
[4]Saunders MJ,Block E,Sorkin A,et al.A bifunctional converter:fluorescein quenching sc Fv/fluorogen activating protein for photostability and improved signal to noise in fluorescence experiments[J].Bioconjug Chem,2014,25:1556-1564
[5]Valm AM,Oldenbourg R,Borisy GG.Multiplexed Spectral Imaging of 120 Different Fluorescent Labels[J].PLoS One,2016,11:e0158495
[6]Sanford L,Palmer A.Recent Advances in Development of Genetically Encoded Fluorescent Sensors[J].Methods Enzymol,2017,589:1-49
[7]Renaud E,Martineau P,Guglielmi L.Solubility Characterization and Imaging of Intrabodies Using GFP-Fusions[J].Methods Mol Biol,2017,1575:165-174
[8]Santos-Beneit F,Errington J.Green fluorescent protein as a reporter for the spatial and temporal expression of actIII in Streptomyces coelicolor[J].Arch Microbiol,2017,199:875-880
[9]Rodriguez-Garcia A,Rojo-Ruiz J,Navas-Navarro P,et al.GAP,an aequorin-based fluorescent indicator for imaging Ca2+in organelles[J].Proc Natl Acad Sci U S A,2014,111:2584-2589
[10]Donaldson JG.Immunofluorescence Staining[J].Curr Protoc Cell Biol,2015,69:431-437
[11]Maity B,Sheff D,Fisher RA.Immunostaining:detection of signaling protein location in tissues,cells and subcellular compartments[J].Methods Cell Biol,2013,113:81-105
[12]Sellars LE,Bryant JA,Sanchez-Romero MA,et al.Development of a new fluorescent reporter:operator system:location of Ara C regulated genes in Escherichia coli K-12[J].BMC Microbiol,2017,17:170
[13]张广峰,陈祥贵,帅培强,等.细胞固定剂对GFP发光特性影响的研究[J].化学与生物工程,2010,27:38-40
[14]Otali D,He Q,Grizzle WE.The effect of antigen retrieval on cells fixed in 10%neutral buffered formalin followed by transfer to 70%ethanol[J].PLo S One,2013,8:e82405
[15]Kuzmin AN,Pliss A,Prasad PN.Changes in biomolecular profile in a single nucleolus during cell fixati on[J].Anal Chem,2014,86:10909-10916
[16]Panda R,Krieger T,Hopf L,et al.Neutrophil Extracellular Traps Contain Selected Antigens of Anti-Neutrophil Cytoplasmic Antibodies[J].Front Immunol,2017,8:439
[17]韩陈陈,马旸,李亦凡,等.不同固定液对内皮细胞形态和EP4荧光染色的影响[J].安徽医科大学学报,2016,51:1372-1374
[18]林哲绚,罗红军,李慧,等.免疫荧光技术中不同固定剂对细胞p65核移位观察效果的影响[J].山西医药杂志,2012,41:211-212
[19]Smith-Clerc J,Hinz B.Immunofluorescence detection of the cytoskeleton and extracellular matrix in tissue and cultured cells[J].Methods Mol Biol,2010,611:43-57
[20]Rogers GL,Hoffman BE.Optimal Immunofluorescent Staining for Human Factor IX and Infiltrating T Cells following Gene Therapy for Hemophilia B[J].J Genet Syndr Gene Ther,2012 Aug 15;S1.pii:012
[21]Yamanushi TT,Boyett MR,Yamamoto Y,et al.Comparison of formaldehyde and methanol fixatives used in the detection of ion channel proteins in isolated rat ventricular myocytes by immunofluorescence labelling and confocal microscopy[J].Folia Morphol(Warsz),2015,74:258-261
[22]Hasegawa Y,Mark Welch JL,Rossetti BJ,et al.Preservation of three-dimensional spatial structure in the gut microbiome[J].PLoSOne,2017,12:e0188257
[23]Rieger J,Twardziok S,Huenigen H,et al.Porcine intestinal mast cells.Evaluation of different fixatives for histochemical staining techniques considering tissue shrinkage[J].Eur J Histochem,2013,57:e21
[24]Pereira MA,Dias AR,Faraj SF,et al.Carnoy's solution is an adequate tissue fixative for routine surgical pathology,preserving cell morphology and molecular integrity[J].Histopathology,2015,66:388-397