免疫荧光染色质量影响因素分析
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摘要
免疫荧光技术是标记免疫技术中应用最为广泛的一项技术,具有特异性强、灵敏度高、速度快和便于观察等特点。影响免疫荧光染色质量的因素多种多样,从荧光染料、抗体、样品前处理、染色过程等几个重要的方面入手,就如何提高免疫荧光染色质量,以获得一张高分辨率的图片做了详细的介绍。该研究有利于提高免疫荧光染色质量,为生命科学研究提供了有力帮助。
As one of the most widely used immunolabeling techniques,immunofluorescence has several advantages including high specificity,high sensitivity,rapid detection and clear positioning. However,the quality of immunofluorescent staining could be affected by multiple factors,such as fluorescent dyes,antibodies,sample preparation and staining process.Starting with those vital factors,we introduced how to improve the quality of immunofluorescent staining so as to obtain a high-resolution image in detail.This study improved the quality of immunofluorescent staining and provided powerful help for life science research.
As one of the most widely used immunolabeling techniques,immunofluorescence has several advantages including high specificity,high sensitivity,rapid detection and clear positioning. However,the quality of immunofluorescent staining could be affected by multiple factors,such as fluorescent dyes,antibodies,sample preparation and staining process.Starting with those vital factors,we introduced how to improve the quality of immunofluorescent staining so as to obtain a high-resolution image in detail.This study improved the quality of immunofluorescent staining and provided powerful help for life science research.
引文
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[16] ISIDRO R A,ISIDRO A A,CRUZ M L,et al.Double immunofluorescent staining of rat macrophages in formalin-fixed paraffin-embedded tissue using two monoclonal mouse antibodies[J].Histochem Cell Biol,2015,144(6):613-621.
[17] MAHMOUDIAN J,HADAVI R,JEDDI-TEHRANI M,et al.Comparison of the photobleaching and photostability traits of Alexa Fluor 568-and fluorescein isothiocyanate- conjugated antibody[J].Cell J,2011,13(3):169-172.
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[19] KAJIMURA J,ITO R,MANLEY N R,et al.Optimization of single- and dual-color immunofluorescence protocols for formalin-fixed,paraffin-embedded archival tissues[J].J Histochem Cytochem,2016,64(2):112-124.
[20] 边玮.激光共聚焦显微镜样品制备方法(二)——组织切片样品[J].电子显微学报,2010,29(4):399-402.
[21] KLAUSS A,K?NIG M,HILLE C.Upgrade of a scanning confocal microscope to a single-beam path STED microscope[J].PLoS One,2015,10(6):1-27.
[22] GOOSSENS K,VANDAELE L,WYDOOGHE E,et al.The importance of adequate fixation for immunofluorescent staining of bovine embryos[J].Reprod Domest Anim,2011,46(6):1098-1103.
[23] NIIKURA Y,KITAGAWA K.Immunofluorescence analysis of endogenous and exogenous centromere-kinetochore proteins[J].J Vis Exp,2016,109:1-16.
[24] LIN C I,LAU C Y,LI D,et al.Nanopodia-thin,fragile membrane projections with roles in cell movement and intercellular interactions[J].J Vis Exp,2014,86:1-6.
[25] BONACCORSI S,GIANSANTI M G,CENCI G,et al.Paraformaldehyde fixation of Drosophila testes[J].Cold Spring Harb Protoc,2012(1):102-104.
[26] LIU H S,KAO W W Y.A novel protocol of whole mount electro-immunofluorescence staining[J].Mol Vis,2009,15:505-517.
[27] SPECTOR D L.Immunofluorescence localization of nuclear proteins[J].Cold Spring Harb Protoc,2011(10):1276-1280.
[28] HARIZANOVA J,FERMIN Y,MALIK-SHERIFF R S,et al.Highly multiplexed imaging uncovers changes in compositional noise within assembling focal adhesions[J].PLoS One,2016,11(8):1-18.
[29] ROSAS-ARELLANO A,VILLALOBOS-GONZáLEZ J B,PALMA-TIRADO L,et al.A simple solution for antibody signal enhancement in immunofluorescence and triple immunogold assays[J].Histochem Cell Biol,2016,146(4):421-430.
[30] HARTMANN D,KRAMMER S,VURAL S,et al.Immunofluorescence and confocal microscopy for ex-vivo diagnosis of melanocytic and non-melanocytic skin tumors:A pilot study[J/OL].J Biophotonics,2018,11(3)[2019-03-20].https://doi.org/10.1002/jbio.201700211.
[31] BUCHWALOW I,SAMOILOVA V,BOECKER W,et al.Non-specific binding of antibodies in immunohistochemistry:Fallacies and facts[J].Sci Rep,2011,1:1-6.
[2] KIVI G,TEESALU K,PARIK J,et al.HybriFree:A robust and rapid method for the development of monoclonal antibodies from different host species[J].BMC Biotechnol,2016,16:1-14.
[3] PAN J,THOENI C,MUISE A,et al.Multilabel immunofluorescence and antigen reprobing on formalin-fixed paraffin-embedded sections:Novel applications for precision pathology diagnosis[J].Mod Pathol,2016,29(6):557-569.
[4] SATPATHY G,BEHERA H S,SHARMA A,et al.A 20-year experience of ocular herpes virus detection using immunofluorescence and polymerase chain reaction[J].Clin Exp Optom,2018,101(5):648-651.
[5] VASQUEZ J J,HUSSIEN R,AGUILAR-RODRIGUEZ B,et al.Elucidating the burden of HIV in tissues using multiplexed immunofluorescence and in situ hybridization:Methods for the single-cell phenotypic characterization of cells harboring HIV in situ[J].J Histochem Cytochem,2018,66(6):427-446.
[6] 赵文晓.掺杂对YAG:Ce3+荧光粉光学性能的影响[D].大连:大连工业大学,2014.
[7]ZHANG D P,XIE C J,WANG R Z,et al.Effective preparation of a monoclonal antibody against human chromogranin A for immunohistochemical diagnosis[J].BMC Biotechnol,2018,18(1):1-9.
[8]李丽.三种人兽共患病病原菌同步快速检测技术研究[D].长春:吉林大学,2017.
[9]TANG X,CHEN J,WANG Y,et al.Gene cloning,expression and polyclonal antibody preparation of Rab3A for protein interaction analysis[J].Springerplus,2016,5(1):1-7.
[10] ZHENG H L,LI H,SUN Y S,et al.Parathyroid hormone-related peptide (PTHrP):Prokaryotic expression,purification,and preparation of a polyclonal antibody[J].Genet Mol Res,2014,13(3):6448-6454.
[11] 程留奇.晚期肺腺癌患者组织中p53、PD-L1表达与预后的关系[D].郑州:郑州大学,2016.
[12] KOMIYAMA N,HIEMORI M,KIMOTO M,et al.Preparation and epitope mapping of a monoclonal antibody against Tri a Bd 27K,a major wheat allergen[J].Biosci Biotechnol Biochem,2009,73(9):2113-2116.
[13] MESSIAS N C,WALKER P D,LARSEN C P.Paraffin immunofluorescence in the renal pathology laboratory:More than a salvage technique[J].Mod Pathol,2015,28(6):854-860.
[14] ZHANG D Y,SUN T Z,YAN J Y,et al.Secretory expression of negative regulatory region of human Notch1 in Escherichia coli and preparation of a functional polyclonal antibody[J].Biotechnol Appl Biochem,2018,65(4):554-559.
[15] BASTIDE A,YEWDELL J W,DAVID A.The RiboPuromycylation Method (RPM):An immunofluorescence technique to map translation sites at the sub-cellular level[J/OL].Bio Protoc,2018,8(1)[2019-04-20].https://bio-protocol.org/e2669.DOI:10.21769/BioProtoc.2669.
[16] ISIDRO R A,ISIDRO A A,CRUZ M L,et al.Double immunofluorescent staining of rat macrophages in formalin-fixed paraffin-embedded tissue using two monoclonal mouse antibodies[J].Histochem Cell Biol,2015,144(6):613-621.
[17] MAHMOUDIAN J,HADAVI R,JEDDI-TEHRANI M,et al.Comparison of the photobleaching and photostability traits of Alexa Fluor 568-and fluorescein isothiocyanate- conjugated antibody[J].Cell J,2011,13(3):169-172.
[18] ZHANG Z Q,IBRAHIM M,FU Y,et al.Application of laser scanning confocal microscopy in the soft tissue exquisite structure for 3D scan[J].Int J Burns Trauma,2018,8(2):17-25.
[19] KAJIMURA J,ITO R,MANLEY N R,et al.Optimization of single- and dual-color immunofluorescence protocols for formalin-fixed,paraffin-embedded archival tissues[J].J Histochem Cytochem,2016,64(2):112-124.
[20] 边玮.激光共聚焦显微镜样品制备方法(二)——组织切片样品[J].电子显微学报,2010,29(4):399-402.
[21] KLAUSS A,K?NIG M,HILLE C.Upgrade of a scanning confocal microscope to a single-beam path STED microscope[J].PLoS One,2015,10(6):1-27.
[22] GOOSSENS K,VANDAELE L,WYDOOGHE E,et al.The importance of adequate fixation for immunofluorescent staining of bovine embryos[J].Reprod Domest Anim,2011,46(6):1098-1103.
[23] NIIKURA Y,KITAGAWA K.Immunofluorescence analysis of endogenous and exogenous centromere-kinetochore proteins[J].J Vis Exp,2016,109:1-16.
[24] LIN C I,LAU C Y,LI D,et al.Nanopodia-thin,fragile membrane projections with roles in cell movement and intercellular interactions[J].J Vis Exp,2014,86:1-6.
[25] BONACCORSI S,GIANSANTI M G,CENCI G,et al.Paraformaldehyde fixation of Drosophila testes[J].Cold Spring Harb Protoc,2012(1):102-104.
[26] LIU H S,KAO W W Y.A novel protocol of whole mount electro-immunofluorescence staining[J].Mol Vis,2009,15:505-517.
[27] SPECTOR D L.Immunofluorescence localization of nuclear proteins[J].Cold Spring Harb Protoc,2011(10):1276-1280.
[28] HARIZANOVA J,FERMIN Y,MALIK-SHERIFF R S,et al.Highly multiplexed imaging uncovers changes in compositional noise within assembling focal adhesions[J].PLoS One,2016,11(8):1-18.
[29] ROSAS-ARELLANO A,VILLALOBOS-GONZáLEZ J B,PALMA-TIRADO L,et al.A simple solution for antibody signal enhancement in immunofluorescence and triple immunogold assays[J].Histochem Cell Biol,2016,146(4):421-430.
[30] HARTMANN D,KRAMMER S,VURAL S,et al.Immunofluorescence and confocal microscopy for ex-vivo diagnosis of melanocytic and non-melanocytic skin tumors:A pilot study[J/OL].J Biophotonics,2018,11(3)[2019-03-20].https://doi.org/10.1002/jbio.201700211.
[31] BUCHWALOW I,SAMOILOVA V,BOECKER W,et al.Non-specific binding of antibodies in immunohistochemistry:Fallacies and facts[J].Sci Rep,2011,1:1-6.