6免疫细胞化学综合应用无

1、免疫细胞化学技术在生物医学科研中的应用易静上海交通大学医学院生化与分子细胞生物学系后基因组时代生物医学研究后基因组时代生物医学研究的常见问题的常见问题l 某个基因的表达情况（在特定的生理、病理条件下；mRNA和蛋白质）l 某个新基因所编码的蛋白质的功能 （在特定组织、细胞、细胞器的分布和相互作用分子）细胞化学，特别是免疫细胞化学技术，细胞化学，特别是免疫细胞化学技术，是用于回答这些问题的最简易的技术是用于回答这些问题的最简易的技术细胞化学：l 酶细胞化学 enzymal cytochemistry（酶-底物）l 免疫细胞化学 immunocytochemistry（抗原-抗体）l 原位杂交 i

2、n situ hybridization（核苷酸-核苷酸探针）什么是细胞化学？什么是细胞化学？检测和分析细胞的化学组成？检测和分析细胞的化学组成？免疫印迹免疫印迹光度测定光度测定什么是细胞化学？什么是细胞化学？在原位在原位检测和分析细胞的化学组成检测和分析细胞的化学组成流式细胞仪流式细胞仪普通光镜普通光镜荧光显微镜荧光显微镜激光共焦显微镜激光共焦显微镜电子显微镜电子显微镜 了解一个基因在组织和细胞里是否表达、了解一个基因在组织和细胞里是否表达、表达水平的高低，除了免疫细胞化学，还可以表达水平的高低，除了免疫细胞化学，还可以用的技术是用的技术是- 免疫细胞化学免疫细胞化学 vs vs 免疫印迹技

3、术免疫印迹技术 的选择的选择 免疫细胞化学免疫细胞化学 vs vs 原位杂交技术原位杂交技术 的选择的选择 原位杂交技术原位杂交技术 vs RT-PCRvs RT-PCR技术技术 的选择的选择lysis免疫印迹免疫组化fixation免疫细胞化学免疫细胞化学 vs 免疫印迹（免疫印迹（Western Blotting）分子量分子量marker分子量分子量marker转录转录翻译翻译免疫细胞化学免疫细胞化学 vs 原位杂交原位杂交DNAmRNAprotein降解降解1.考虑基因表达调控环节，分析mRNA和蛋白质分别反映的信息2. 考虑mRNA和蛋白质各自的动力学，预测定位mRNA位于细胞质pro

4、tein位于细胞质 细胞核 细胞膜 细胞外lysisRT-PCR原位杂交fixation原位杂交技术原位杂交技术 vs RT-PCRRT+PCR分子量分子量marker免疫细胞化学(immunocytochemistry) 技术原理 抗原（待检分子） 标记抗体 或或 标记的抗原抗体复合物 抗体标记系统标记的抗原抗体复合物 显色物质观察、检测原位杂交(in situ hybridization) 技术原理标记的抗原抗体复合物 显色物质观察、检测 核苷酸（待检分子）+ 标记的核苷酸探针 标记的杂交体 抗原为抗原 标记抗体 或或 标记的抗原抗体复合物 抗体标记系统终产物是使抗体或抗原抗体复合物在仪器

5、下终产物是使抗体或抗原抗体复合物在仪器下 可见或可探测的物质。可见或可探测的物质。 （一）（一） 免疫荧光技术免疫荧光技术-将荧光素作为标记物（二）（二） 免疫酶技术和亲和细胞化学技术免疫酶技术和亲和细胞化学技术- 将酶（HRP & AKP）作为抗原抗体复合物的标记物，再通过酶细胞化学反应产生在光镜下可见的显色物质或电镜下可见的高电子密度物质 （三）（三） 免疫胶体金技术免疫胶体金技术-用金标抗体或金标A蛋白作为二抗，反应部位在电镜下为高电子密度的金颗粒所指示。 间接法间接法 直接法直接法免疫细胞化学：免疫细胞化学：直接和间接两种方法直接和间接两种方法免疫细胞细胞化学 (immunoc

6、ytochemistry, ICC)免疫组织组织化学 (immunohistochemistry, IHC)vs两个名词可互相取代使用，但常分别用于不同样品培养细胞或组织分散细胞组织A human colon carcinoma tissue section (IHC) immunocytochemistry (ICC) and immunohistochemistry (IHC) stainingA549 human adenocarcinoma cells (ICC) Samples were fixed and stained for protein phosphatase 2 (PP2

7、A) by indirect detection using Horseradish Peroxidase (HRP)-Conjugated Goat Anti-Mouse Secondary Antibody targeting the anti-PP2A primary antibody. Enzymatic development was completed using Pierce Metal-Enhanced DAB.Immunocytochemistry (ICC) in and immunohistochemistry (IHC) in Immunocytochemistry I

8、mmunohistochemistry Immunohistochemistry and microRNA in situ hybridization performed on the tissue section. Cerebellum and hippocampus of a mouse brain are visualized. Red colour shows GFAP protein which is found in neuronal cells, green colour shows miRNA expression while the blue colour shows a n

9、uclear staining. Combine immunohistochemical detection of protein/peptide biomarkers and in situ hybridization of nucleic acid on the same tissue section 光镜和电镜水平的免疫细胞化学技术 综合应用，主要解决的问题：特异蛋白质的定位特异蛋白质的定位 1. 大分子的组织和细胞水平定位 tissue and cellular localization 2. 细胞内大分子的固定亚细胞定位 subcellular localization 3. 细胞内

10、大分子的转运或移位 translocation 4. 细胞内定位固定的大分子的动态变化 dynamics 5. 细胞内大分子的共定位 co-localization 大分子的组织和细胞水平定位 tissue and cellular localization 0481216SENPpcDNA3Relative signal intensity Positive area(%)0204060SENPpcDNA3CD31SENPpcDNA3VEGFISH normal colon tissue colon adenocarcinomaAimmunohistochemistry Bcolonrect

11、umprostateovariumlungesophagusgastrokidney-1001020304050607080Positive area ratio%normal tissuecancer tissuepancreasN= 16, 32 20, 65 9, 10 8, 8 3, 3 3, 3 3, 3 3, 3 3, 3移植瘤病人标本IHC Huang C. Han Y. et al. EMBO J. 2009 pcDNA3RGS-SENP3AB Han Y. et al. JBC. 2010 我们的工作我们的工作SENP3蛋白在多种人类肿瘤中水平上调，通过促进HIF-1活性发挥

12、促癌作用病人垂体腺瘤标本 Yi J. et al. Chin Med J. 2000 我们的工作我们的工作p16蛋白在垂体腺瘤细胞中水平各异，总体上高于间质细胞，而其mRNA水平均等P16 蛋白蛋白（IHC）P16 mRNA（ISH）大分子亚细胞水平定位 subcellular localization ABC法显示法显示型胶原在病变肾小球中的分布型胶原在病变肾小球中的分布电镜胶体金法显示肾小球基底膜上的电镜胶体金法显示肾小球基底膜上的型胶原蛋白型胶原蛋白我们的工作我们的工作(汤雪明，未发表材料)电镜原位杂交胶体金法显示肾小血管外膜成纤维细胞的I型胶原蛋白mRNA我们的工作我们的工作易静， 1

13、995 (J Histochem Cytochem)海马细胞 微管蛋白 绿色(胞体、树突) 轴突终末蛋白 红色(突触) 黄色？ Neurobiology突触 synaps李瑞锡等. J Comp Neurol. 2001 Oct 29;439(4):411-25.Light and electron microscopic study of cholinergic and noradrenergic elements in the basolateral nucleus of the rat amygdala: evidence for interactions between the two

14、 systems.等等免疫细胞化学技术（免疫荧光）免疫细胞化学技术（免疫荧光）: : 近年大量用于表达重组蛋白的培养细胞近年大量用于表达重组蛋白的培养细胞目的在于：了解功能未知的新蛋白亚细胞定位 subcellular localization多分子共定位 co-localizationIEM1. To investigate the subcellular localization2. To validate the organelle proteomic results subcellular localization带标签的重组带标签的重组DNADNARecombinant DNAtag

15、tagco-localizationPNAS 2002SIRT3 Protein Is Targeted to the Mitochondria. As a clue to the function of SIRT3, we investigated the subcellular location of the protein. Fig. 4 shows that the SIRT3 protein is localized to the mitochondria.SIRT3, a human SIR2 homologue, is an NAD-dependent deacetylase l

16、ocalized to mitochondriaThese results suggest a previously unrecognized organelle for sirtuin function and that the role of SIRT3 in mitochondria involves protein deacetylation.Fig. 3. Cellular localization of SIRT3 full-length and truncated proteins in Cos7 cells. (A)Shown are cells transfected wit

17、h vector plasmid. Strong expression of the EGFP in the nucleus and also expression in the cytoplasm is evident. Nuclei were stained with DAPI and colored red, hence the yellow coloration when the green and red signals are superimposed in the nucleus. (B) The N-terminal (1142 aa) deleted SIR2T3 gene

18、fused in-frame to the EGFP was transfected. Note the diffused expression of the chimeric EGFP protein throughout the cell. Unlike with the EGFP shown above, there was no preferential expression of the chimeric protein in the nucleus. (C) SIR2T3 fused to EGFP (green), localized predominantly around t

19、he nuclear periphery. Red shows the nucleus detected by counterstaining with DAPI .空载：核为主短SIRT3：弥散长SIRT3：细胞质，细胞器Fig. 4. Subcellular localization of the full-length SIRT3 gene in Cos7 cells. A shows DAPI staining of the nuclei,and B depicts the localization of the SIRT3-EGFP protein. Shown in red in

20、C is the mitochondrial marker, Mitotracker. D shows superimposed images of AC. It is clear from D that SIRT3 localizes to the mitochondria.MitotrackerSIRT3-EGFPDAPIFig. 6. Immunoelectron microscopy showing localization of SIRT3EGFP to the mitochondrial cristae. (A) Indirect immunogold labeling of ul

21、trathin cryosections of human cervical carcinoma (SiHa) cells demonstrates SIRT3EGFP label (5 nm gold) in plain association with mitochondria in three independent images (AC).High-magnification images reveal label clearly associated with mitochondrial cristae (B and C). m, mitochondria; arrowheads,

22、cristae labeling. (Bars, 0.5 m.)LC3 localizes on the membranes of autophagosomes细胞内大分子的转运或移位 translocationIEMOrganells-organelles (Vesicles-vesicles)LMCytoplasma-nucleusOrganells-organelles (organelle-track dyes)Organells-cytosol (organelle-track dyes)易静研究组，杨凯，未发表氧化应激下核仁蛋白氧化应激下核仁蛋白X移位到核质移位到核质内源蛋白免疫荧

23、光XDICDAPIControlH2O2我们的工作我们的工作33000-+012345核仁颗粒密度/核质颗粒密度*H2O2包埋后免疫电镜胶体金技术标记核仁蛋白XctrlH2O2 500uM核仁核质*金颗粒我们的工作我们的工作易静研究组，杨凯，未发表我们的工作我们的工作单细胞单分子荧光示踪技术排查核仁蛋白X负责应激下移位的半胱氨酸残基易静研究组，杨凯，未发表细胞内定位固定的大分子的动态变化 dynamics IEM To demonstrate the appearance and disappearance of the proteins on the vesicular membrane 蛋

24、白质出现或消失，往往反映蛋白质降解改变（与转位translocation的区别）核仁蛋白SENP3在轻度氧化应激条件下在核质中累积Huang C. Han Y. et al. EMBO J. 2009, 我们的工作我们的工作Clathrin出现在高尔基体反面管网结构刚刚芽生形成的分泌小泡膜上，但在内含物已经发生浓缩的成熟小泡膜上消失 Orci.L et al. Cell, 1987clathrinclathrinDe-coating三种类型的有被小泡介导不同的运输途径三种类型的有被小泡介导不同的运输途径教科书内容：Molecular Biology of the Cell B.Alberts

25、et al 2008细胞内大分子的共定位 co-localizationProtein-protein interaction IEM To validate the co-IP results To confirm the subcellular co-localization 两种方法 Dual staining （multi-staining） FRET-acceptor photobleaching fluorescence resonance energy transfer (FRET). 1. based on immunohistochemistry 2. fluorescent

26、 protein vectors transfectionFRET-acceptor photobleaching fluorescence resonance energy transfer (FRET).bleachingclassicalAcceptor photobleachingNature，2005.光镜和电镜的双标记免疫细胞化学：光镜和电镜的双标记免疫细胞化学： 两个蛋白质分子的共定位lHere, we show that clathrin stabilizes fibres of the mitotic spindle to aid congression of chromos

27、omes. Stabilization of kinetochore fibres was dependent on the unique structure of clathrin. lThe importance of clathrin for normal mitosis may be relevant to understanding human cancers that involve gene fusions of clathrin heavy chain.Clathrin has an established function in the generation of vesic

28、les.The formation of clathrin-coated vesicles occurs continuously in nondividing cells, but is shut down during mitosis, when clathrin concentrates at the spindle apparatus.Figure 1 Clathrin was targeted to the mitotic spindle of NRK cells. a, Confocal micrographs showing the subcellular distributio

29、n of clathrin at interphase and metaphase. GFPLCa (left, green), a-tubulin (centre, red) and nucleic acid (blue) staining is shown.b, Cells expressing GFPLCa fixed before (top) or after (bottom) cold treatment to depolymerize non-kinetochore microtubules. CENPB, centromere protein B. c, d, The assoc

30、iation of clathrin with microtubules is not via coated membranes. c, Example images of live cells expressing either GFPa-tubulin (left) or GFPLCa (right) imaged after 2428-h incubation with FM4-64 (red). d, Association of clathrin with microtubules visualized by immunogold electron microscopy. CHC (

31、15 nm gold) and a-tubulin (10 nm gold) in mitotic NRK cells.a, Confocal micrographs showing the subcellular distribution of clathrin at interphase and metaphase.GFPLCa (left, green),a-tubulin (centre, red) and nucleic acid (blue) staining is shown.b, Cells expressing GFPLCa fixed before (top) or aft

32、er (bottom) cold treatment to depolymerize non-kinetochore microtubules. CENPB, centromere protein B.c, d, The association of clathrin with microtubules is not via coated membranes. c, Example images of live cells expressing either GFPa-tubulin (left) or GFPLCa (right) imaged after 2428-h incubation

33、 with FM4-64 (red).To test whether clathrin at the spindle was associated with membranes at all, we indiscriminately labelled intracellular compartments by incubating cells with the styryl dye FM4-64 (15mM) for .24 h. In cells at metaphase, none of these membranes was found at the spindle (Fig. 1c).

34、c, d, The association of clathrin with microtubules is not via coated membranes. d, Association of clathrin with microtubules visualized by immunogold electron microscopy. CHC (15 nm gold) and a-tubulin (10 nm gold) in mitotic NRK cells. Chromosomes are denoted by asterisks. A morphologically distin

35、ct clathrin-coated vesicle (bottom left panel) is indicated by an arrow. Arrowheads denote CHC labelling associated with microtubules.Role of neuropeptide Y in the regulation of gonadotropin releasing hormone system in the forebrain of Clarias batrachus (Linn.): . Neuroscience. 2005;133(1):267-79The

36、 present investigation provided novel information on the extensive anatomical association of the NPY and GnRH containing systems in the forebrain. The overlapping of the two peptide containing systems seems to provide the neuroanatomical substrate for NPY to exercisepositive control over GnRHNPY con

37、taining axons were found to terminate in the vicinity of GnRH cells in the pituitary. Double immunoelectron microscopy demonstrated gold particles for NPY (40nm) and GnRH (10nm) colocalized on the membrane and in dense core of the secretory granules in the cells distributed in all components of the pituitary gland.FIG. 1. Confocal imaging of EGFR phosphorylation in a SW-480 cell by acceptor photobleaching FRET. a, F4-Cy3 stainingbefore photobleaching. b, Py72-Cy5 staining before photobleaching. c, F4-Cy3 staining after photobleaching of Cy5. The area in which Cy5 was photobleach