免疫細(xì)胞化學(xué)綜合應(yīng)用課件

1、免疫細(xì)胞化學(xué)技術(shù)在生物醫(yī)學(xué)科研中的應(yīng)用后基因組時(shí)代生物醫(yī)學(xué)研究的常見(jiàn)問(wèn)題某個(gè)基因的表達(dá)情況（在特定的生理、病理?xiàng)l件下；mRNA和蛋白質(zhì)）某個(gè)新基因所編碼的蛋白質(zhì)的功能 （在特定組織、細(xì)胞、細(xì)胞器的分布和相互作用分子）博士生入學(xué)考試題目之一體外研究發(fā)現(xiàn)某基因具有促進(jìn)腫瘤細(xì)胞生長(zhǎng)的作用，你認(rèn)為應(yīng)采用哪些策略開(kāi)展進(jìn)一步研究，以明確該基因確實(shí)與人類腫瘤的發(fā)生發(fā)展有關(guān)？細(xì)胞化學(xué)，特別是免疫細(xì)胞化學(xué)技術(shù)，是用于回答這些問(wèn)題的最簡(jiǎn)易的技術(shù)細(xì)胞化學(xué)：酶細(xì)胞化學(xué) enzymal cytochemistry（酶-底物）免疫細(xì)胞化學(xué) immunocytochemistry（抗原-抗體）原位雜交 in situ hy

2、bridization（核苷酸-核苷酸探針）什么是細(xì)胞化學(xué)？檢測(cè)和分析細(xì)胞的化學(xué)組成？免疫印跡光度測(cè)定什么是細(xì)胞化學(xué)？在原位檢測(cè)和分析細(xì)胞的化學(xué)組成流式細(xì)胞儀普通光鏡熒光顯微鏡激光共焦顯微鏡電子顯微鏡 了解一個(gè)基因在組織和細(xì)胞里是否表達(dá)、表達(dá)水平的高低，除了免疫細(xì)胞化學(xué)，還可以用的技術(shù)是-免疫細(xì)胞化學(xué) vs 免疫印跡技術(shù) 的選擇免疫細(xì)胞化學(xué) vs 原位雜交技術(shù) 的選擇原位雜交技術(shù) vs RT-PCR技術(shù) 的選擇lysis免疫印跡免疫組化fixation免疫細(xì)胞化學(xué) vs 免疫印跡（Western Blotting）分子量marker分子量marker轉(zhuǎn)錄翻譯免疫細(xì)胞化學(xué) vs 原位雜交DNAm

3、RNAprotein降解1.考慮基因表達(dá)調(diào)控環(huán)節(jié)，分析mRNA和蛋白質(zhì)分別反映的信息2. 考慮mRNA和蛋白質(zhì)各自的動(dòng)力學(xué)，預(yù)測(cè)定位mRNA位于細(xì)胞質(zhì)protein位于細(xì)胞質(zhì) 細(xì)胞核 細(xì)胞膜 細(xì)胞外lysisRT-PCR原位雜交fixation原位雜交技術(shù) vs RT-PCRRT+PCR分子量marker免疫細(xì)胞化學(xué)(immunocytochemistry) 技術(shù)原理 抗原（待檢分子） 標(biāo)記抗體 或 標(biāo)記的抗原抗體復(fù)合物 抗體標(biāo)記系統(tǒng)標(biāo)記的抗原抗體復(fù)合物 顯色物質(zhì)觀察、檢測(cè)原位雜交(in situ hybridization) 技術(shù)原理標(biāo)記的抗原抗體復(fù)合物 顯色物質(zhì)觀察、檢測(cè) 核苷酸（待檢分子

4、）+ 標(biāo)記的核苷酸探針 標(biāo)記的雜交體 抗原為抗原 標(biāo)記抗體 或 標(biāo)記的抗原抗體復(fù)合物 抗體標(biāo)記系統(tǒng)終產(chǎn)物是使抗體或抗原抗體復(fù)合物在儀器下 可見(jiàn)或可探測(cè)的物質(zhì)。 （一） 免疫熒光技術(shù)-將熒光素作為標(biāo)記物（二） 免疫酶技術(shù)和親和細(xì)胞化學(xué)技術(shù)- 將酶（HRP & AKP）作為抗原抗體復(fù)合物的標(biāo)記物，再通過(guò)酶細(xì)胞化學(xué)反應(yīng)產(chǎn)生在光鏡下可見(jiàn)的顯色物質(zhì)或電鏡下可見(jiàn)的高電子密度物質(zhì) （三） 免疫膠體金技術(shù)-用金標(biāo)抗體或金標(biāo)A蛋白作為二抗，反應(yīng)部位在電鏡下為高電子密度的金顆粒所指示。 間接法 直接法免疫細(xì)胞化學(xué)：直接和間接兩種方法免疫細(xì)胞化學(xué) (immunocytochemistry, ICC)免疫組織化學(xué) (

5、immunohistochemistry, IHC)vs兩個(gè)名詞可互相取代使用，但常分別用于不同樣品培養(yǎng)細(xì)胞或組織分散細(xì)胞組織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 (PP2A) by indirect detection usi

6、ng 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 ImmunohistochemistryImmunohis

7、tochemistry 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 nuclear staining. Combine immu

8、nohistochemical detection of protein/peptide biomarkers and in situ hybridization of nucleic acid on the same tissue section 光鏡和電鏡水平的免疫細(xì)胞化學(xué)技術(shù) 綜合應(yīng)用，主要解決的問(wèn)題：特異蛋白質(zhì)的定位1. 大分子的組織和細(xì)胞水平定位 tissue and cellular localization2. 細(xì)胞內(nèi)大分子的固定亞細(xì)胞定位 subcellular localization3. 細(xì)胞內(nèi)大分子的轉(zhuǎn)運(yùn)或移位 translocation4. 細(xì)胞內(nèi)定位固定的大分子的動(dòng)態(tài)

9、變化 dynamics 5. 細(xì)胞內(nèi)大分子的共定位 co-localization 大分子的組織和細(xì)胞水平定位 tissue and cellular localization 0481216SENPpcDNA3Relative signal intensity Positive area(%)0204060SENPpcDNA3CD31SENPpcDNA3VEGFISH normal colon tissue colon adenocarcinomaAimmunohistochemistry Bcolonrectumprostateovariumlungesophagusgastrokidne

10、y-1001020304050607080Positive area ratio%normal tissuecancer tissuepancreasN= 16, 32 20, 65 9, 10 8, 8 3, 3 3, 3 3, 3 3, 3 3, 3移植瘤病人標(biāo)本IHC Huang C. Han Y. et al. EMBO J. 2009 pcDNA3RGS-SENP3AB Han Y. et al. JBC. 2010 我們的工作SENP3蛋白在多種人類腫瘤中水平上調(diào)，通過(guò)促進(jìn)HIF-1活性發(fā)揮促癌作用病人垂體腺瘤標(biāo)本 Yi J. et al. Chin Med J. 2000 我們的

11、工作p16蛋白在垂體腺瘤細(xì)胞中水平各異，總體上高于間質(zhì)細(xì)胞，而其mRNA水平均等P16 蛋白（IHC）P16 mRNA（ISH）大分子亞細(xì)胞水平定位 subcellular localization ABC法顯示型膠原在病變腎小球中的分布電鏡膠體金法顯示腎小球基底膜上的型膠原蛋白我們的工作(湯雪明，未發(fā)表材料)電鏡原位雜交膠體金法顯示腎小血管外膜成纖維細(xì)胞的I型膠原蛋白mRNA我們的工作易靜， 1995 (J Histochem Cytochem)海馬細(xì)胞 微管蛋白 綠色(胞體、樹(shù)突) 軸突終末蛋白 紅色(突觸) 黃色？ Neurobiology突觸 synaps李瑞錫等. J Comp Ne

12、urol. 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 systems.等大鼠睪丸免疫組化發(fā)現(xiàn)某蛋白的特殊定位核膜？核纖層？上海交通大學(xué) 喬中東教授頂體！免疫細(xì)胞化學(xué)技術(shù)（免疫熒光）: 近年大量用于表達(dá)重組蛋白的培養(yǎng)細(xì)胞目的在于：了解功能未知的新蛋白亞細(xì)

13、胞定位 subcellular localization多分子共定位 co-localizationIEMTo investigate the subcellular localizationTo validate the organelle proteomic results subcellular localization帶標(biāo)簽的重組DNARecombinant DNAtagtagco-localizationPNAS 2002SIRT3 Protein Is Targeted to the Mitochondria. As a clue to the function of SIRT3,

14、 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 localized to mitochondriaThese results suggest a previously unrecognized organelle for sirtuin function and th

15、at 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 with vector plasmid. Strong expression of the EGFP in the nucleus and also expression in the cytoplasm is eviden

16、t. 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 fused in-frame to the EGFP was transfected. Note the diffused expression of the chimeric EGFP protein through

17、out 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 the nuclear periphery. Red shows the nucleus detected by counterstaining with DAPI .空載：核為主短SIRT3：彌散長(zhǎng)SIRT3：細(xì)胞質(zhì)，

18、細(xì)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 C is the mitochondrial marker, Mitotracker. D shows superimposed images of AC. It is clear from D that SIRT3

19、localizes to the mitochondria.MitotrackerSIRT3-EGFPDAPIFig. 6. Immunoelectron microscopy showing localization of SIRT3EGFP to the mitochondrial cristae. (A) Indirect immunogold labeling of ultrathin cryosections of human cervical carcinoma (SiHa) cells demonstrates SIRT3EGFP label (5 nm gold) in pla

20、in association with mitochondria in three independent images (AC).High-magnification images reveal label clearly associated with mitochondrial cristae (B and C). m, mitochondria; arrowheads, cristae labeling. (Bars, 0.5 m.)LC3 localizes on the membranes of autophagosomes細(xì)胞內(nèi)大分子的轉(zhuǎn)運(yùn)或移位 translocationIEM

21、Organells-organelles (Vesicles-vesicles)LMCytoplasma-nucleusOrganells-organelles (organelle-track dyes)Organells-cytosol (organelle-track dyes)易靜研究組，楊凱，未發(fā)表氧化應(yīng)激下核仁蛋白X移位到核質(zhì)內(nèi)源蛋白免疫熒光XDICDAPIControlH2O2我們的工作33000*H2O2包埋后免疫電鏡膠體金技術(shù)標(biāo)記核仁蛋白XctrlH2O2 500uM核仁核質(zhì)*金顆粒我們的工作易靜研究組，楊凱，未發(fā)表我們的工作單細(xì)胞單分子熒光示蹤技術(shù)排查核仁蛋白X負(fù)責(zé)應(yīng)激下移

22、位的半胱氨酸殘基易靜研究組，楊凱，未發(fā)表細(xì)胞內(nèi)定位固定的大分子的動(dòng)態(tài)變化 dynamics IEMTo demonstrate the appearance and disappearance of the proteins on the vesicular membrane蛋白質(zhì)出現(xiàn)或消失，往往反映蛋白質(zhì)降解改變（與轉(zhuǎn)位translocation的區(qū)別）核仁蛋白SENP3在輕度氧化應(yīng)激條件下在核質(zhì)中累積Huang C. Han Y. et al. EMBO J. 2009, 我們的工作Clathrin出現(xiàn)在高爾基體反面管網(wǎng)結(jié)構(gòu)剛剛芽生形成的分泌小泡膜上，但在內(nèi)含物已經(jīng)發(fā)生濃縮的成熟小泡膜上消

23、失 Orci.L et al. Cell, 1987clathrinclathrinDe-coating三種類型的有被小泡介導(dǎo)不同的運(yùn)輸途徑教科書(shū)內(nèi)容：Molecular Biology of the Cell B.Alberts et al 2008細(xì)胞內(nèi)大分子的共定位 co-localizationProtein-protein interaction IEMTo validate the co-IP resultsTo confirm the subcellular co-localization 兩種方法Dual staining （multi-staining）FRET-accept

24、or photobleaching fluorescence resonance energy transfer (FRET). 1. based on immunohistochemistry2. fluorescent protein vectors transfectionFRET-acceptor photobleaching fluorescence resonance energy transfer (FRET).bleachingclassicalAcceptor photobleachingNature，2005.光鏡和電鏡的雙標(biāo)記免疫細(xì)胞化學(xué)： 兩個(gè)蛋白質(zhì)分子的共定位Here

25、, we show that clathrin stabilizes fibres of the mitotic spindle to aid congression of chromosomes. Stabilization of kinetochore fibres was dependent on the unique structure of clathrin. The importance of clathrin for normal mitosis may be relevant to understanding human cancers that involve gene fu

26、sions of clathrin heavy chain.Clathrin has an established function in the generation of vesicles.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

27、the mitotic spindle of 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 after (bottom) cold treatme

28、nt 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 with FM4-64 (red). d,

29、Association of clathrin with microtubules visualized by immunogold electron microscopy. CHC (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) a

30、nd 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 association of clathrin with microtubules is not via coated membranes. c, Example images of live

31、 cells expressing either GFPa-tubulin (left) or GFPLCa (right) imaged after 2428-h incubation 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)

32、for .24 h. In cells at metaphase, none of these membranes was found at the spindle (Fig. 1c).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

33、nm gold) in mitotic NRK cells. Chromosomes are denoted by asterisks. A morphologically distinct 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 hormon

34、e system in the forebrain of Clarias batrachus (Linn.): . Neuroscience. 2005;133(1):267-79The 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

35、to provide the neuroanatomical substrate for NPY to exercisepositive control over GnRHNPY containing 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 a

36、nd in dense core of the secretory granules in the cells distributed in all components of the pituitary gland.特異蛋白質(zhì)的定位localization1.大分子的組織和細(xì)胞水平定位 tissue and cellular localization2. 細(xì)胞內(nèi)大分子的固定亞細(xì)胞定位 subcellular localization3. 細(xì)胞內(nèi)大分子的轉(zhuǎn)運(yùn)或移位 translocation4. 細(xì)胞內(nèi)定位固定的大分子的動(dòng)態(tài)變化 dynamics 5. 細(xì)胞內(nèi)大分子的共定位 co-localization 謝 謝！FIG. 1. Confocal imaging of EGFR phosphorylation in a SW-480 cell by acceptor photobleaching FRET. a, F4-Cy