英文免疫组化幻灯片.ppt

《英文免疫组化幻灯片.ppt》由会员分享，可在线阅读，更多相关《英文免疫组化幻灯片.ppt（21页珍藏版）》请在淘文阁 - 分享文档赚钱的网站上搜索。

1、英文免疫组化第1页，共21页，编辑于2022年，星期二ProcedureIncubate tissue with Ab so it binds to antigen present in the tissueWash tissue so only bound antibody remainsExpose tissue with Ab to second Ab which recognizes 1st Ab2nd Ab is conjugated to fluorescent chemical or enzyme system which forms a visible reaction pro

2、duct第2页，共21页，编辑于2022年，星期二Major GoalMaximize signal(positive staining)Minimize background staining(false positive staining)第3页，共21页，编辑于2022年，星期二Procedure ModificationAdd Neutravidin which will be attracted to biotinylated secondary Ab.Neutravidin will be conjugated to the fluorescent tag Alexa 488(gr

3、een fluorescent tag)or CY(red fluorescent tag)第4页，共21页，编辑于2022年，星期二Ab with Specific Recognition CharacteristicsPrimary Ab raised in rabbitSecondary Ab raised against rabbit serum in some other animal(sheep,goat,or rodents)第5页，共21页，编辑于2022年，星期二Double LabelingFor double labeling-primary Ab rasied in d

4、ifferent animals rabbit and rats in case of double labelingsecondary Ab have different materials conjugated(anti-rabbit IgG conjugated with fluorescein and anti-rat IgG conjugated with HRP)第6页，共21页，编辑于2022年，星期二Tissue Antigens2nd Ab1st Ab2nd Ab conjugated with either fluorescent or enzyme markerHRP o

5、r Alkaline Phosphatase第7页，共21页，编辑于2022年，星期二Detailed ProcedureTissue fixedTissue cut into thinner sections(15uM)Mounted on gelatin or poly-D-lysine sectionsUse of Triton XUse of serums(directed against 2nd Ab),dry milk products,avidin,H2O2第8页，共21页，编辑于2022年，星期二ControlsBest procedure-incubate primary A

6、b with antigen-antigen preabsorptionOmit the primary Ab in the 1st incubation stepUse a positive control-use tissue known to contain antigen of interest.第9页，共21页，编辑于2022年，星期二High Background Staining1.Endogenous peroxidase activity-not completely blocked.2.Non-specific binding of protein to specimen-

7、need for blockers.3.Excessive application of tissue adhesive4.Inadequate rinsing of slides5.Improper antibody dilution6.Drying out of tissue.7.Overdevelopment of substrate.第10页，共21页，编辑于2022年，星期二Possible Causes of Negative StainingSteps for staining not performed in correct sequence.Either primary or

8、 secondary antibody incubation steps were skipped.Destruction of labile antigensImproper fixation and or processing of sample第11页，共21页，编辑于2022年，星期二Possible Causes for Weak StainingSpecimen retains too much liquid after rinsing stepsIncubation times are too short or antibody solution is too weak.Poor

9、 titer of primary antibodyImproper substrate preparation or use of old substrate solution第12页，共21页，编辑于2022年，星期二AntibodiesAb-proteins of the immunoglobulin family that bind to different antigen molecules.Immunoglobulin classes(IgG,IgM,IgE)and subclasses(IgG1,IgG2)Ab are produced by B-lymphocytes of t

10、he immune system.Each clone of B-lymphocytes is programmed to produce a single species of antibody with single specificity for antigen.第13页，共21页，编辑于2022年，星期二Polyclonal Abs contAbs are molecule specificInject animals with pure foreign molecule(not usually seen by the immune system of the animal),anim

11、al produces large amounts of antibody to the molecule.Remove blood from immunized animal and separate and purify form other antibodies.第14页，共21页，编辑于2022年，星期二Polyclonal AntibodiesPolyclonal refers to the fact that many different antibodies may be raised to different parts of the molecule.Each antibod

12、y is produced by a single cell.Many different antibodies are produced to a single molecule and these have come each from different cell clones第15页，共21页，编辑于2022年，星期二Procedure for Polyclonals AbsInject into rabbits a purified molecule such as a protein or peptides shorter lengths)can be synthesized an

13、d injected.Inject rabbit several times over 2-3 months.Separate the antibody of interest using synthetic peptides placed in a column and eluding them off the column.第16页，共21页，编辑于2022年，星期二Polyclonal AntibodiesAbs are molecules produced by immune system against invading microorganisms.Each antibody is

14、 produced by a single cell and the body induces division of these cells to increase Ab production(clones of original cell).Once antibodies are bound to invading organism,specified cells destroy the antibody-bound microbe.第17页，共21页，编辑于2022年，星期二Monoclonal AntibodiesMabs are produced by fusion of an im

15、mortalized myeloma(cancer)cell line with a B-lymphocyte to produce a hydridoma cell line.Once you isolate the hybridoma clone with the desired antigen specificity,it is possible to produce an endless supply of highly specific antibody.第18页，共21页，编辑于2022年，星期二Advantages of MabsKey advantage-specificity

16、Polyclonal are collected from serum of immunized animals and contain a mixture of antibody species that recognize different substructure(epitopes)within the target antigen molecule Another advantage-quantitySupply of polyclonal antibodies are limited to amount of serum第19页，共21页，编辑于2022年，星期二Sources o

17、f MabsRepositories include American Type Culture Collectionhttp:/www.atcc.org/catalogs.htmlCommerical suppliers$200-$500/20ugMSRS Catalog claims to provide a comprehensive inventory of all commercially available antibodies第20页，共21页，编辑于2022年，星期二Pitfalls in Using MabsKey advantage-highly specific probeBinds to a single epitope An epitope can consist of only 5 aa residues or just a sugar or lipid modification which could be shared by other macromolecules第21页，共21页，编辑于2022年，星期二