免疫組化原位雜交課件

免疫組織化學(xué)和原位雜交ImmunohistochemistryInsituhybridization免疫組織化學(xué)前提條件:所使用的抗體必須能用于免疫組化基本原理:Pathoger利用抗體(antibody)與相應(yīng)抗原(antigen)的特antigen異性結(jié)合,通過與抗體連接的酶反應(yīng)或熒光染料顯示抗原所在的位置Antibodyoww.scienceaid,net常用于標(biāo)記抗體的標(biāo)記物)酶(enzyme)1.辣根過氧化物酶(horseradishperoxidase,HRP)辣根過氧化物酶的作用物(底物,substrate)1)二氨基聯(lián)苯胺(3,3′-diaminobenzidine,DAB)顯色特點(diǎn):棕色不溶于水及脂類溶劑(酒精ethanol,二甲苯xylene)不擴(kuò)散一永久保存2)3氨基9乙基卡巴唑(3-Amino9Ethylcarbazole,AEC)顯色特點(diǎn)一棕紅色不溶于水,但溶于脂類溶劑容易擴(kuò)散不能永久保存3)4氯1萘酚(4-Chloro-1-naphthol,CN)顯色特點(diǎn)藍(lán)色不溶于水,但溶于脂類溶劑容易擴(kuò)散不能永久保存4)四甲基聯(lián)苯胺(3,3,5,5-Tetramethylbenzidine,TMB)顯色特點(diǎn):深藍(lán)色不溶于水及脂類溶劑不擴(kuò)散永久保存2.堿性磷酸酶(alkalinephosphatase,AP)堿性磷酸酶的作用物(底物,substrate)1)四唑淡藍(lán)/⑤5-溴-4-氯-3-吲哚-磷酸鹽(Nitro-BlueˉTetrazolium/5-bromo-4-chloro-3-inodly1phosphate,NBT/BCIP)顯色特點(diǎn):紫色—不溶于水但溶于脂類溶劑容易擴(kuò)散不能永久保存2)快紅(固紅/萘酚磷酸鹽,Fastredtr/NaphtholAS-MX)顯色特點(diǎn):桃紅色不溶于水但溶于脂類溶劑容易擴(kuò)散不能永久保存3)快藍(lán)(固藍(lán)BB/萘酚磷酸鹽,Fastbluebb/Naphtholas-MX)顯色特點(diǎn):藍(lán)色不溶于水但溶于脂類溶劑容易擴(kuò)散不能永久保存4)品紅(品紅/萘酚磷酸鹽,Fuchsin/NaphtholAS-TR)顯色特點(diǎn):紅色一不溶于水及脂類溶劑—不擴(kuò)散永久保存DDABAECFastred二)熒光染料(fluorescentdye)ProbeEx(nm)Em(nm)MWNotesReactiveandconjugatedprobesAminocoumarin330SuccinimidylesterMethoxycoumarin317SuccinimidylesterCascadeblue596HydrazidePacificBlue40455406MaleimidePacificOrangeNBD466294NBD-XPhycoerythrin480,565240k480565650iKacychrome,R670,Tri-Color,conjugatesQuantumRedconjugates480,565:743767Red613PE-TexasRed490PeridininchlorphyllproteinTruEd490.675695PerCP-Cy55conjugateFluor494587(GEHealthcareFluorescein495519FITC;pHsensitiveBODIPY-FL503TRITC54757244TRITCX-Rhodamine570576548XRITCLissamine570590RhodamineBRed589615625sulfonylchlorideAllophycocyanin650660104k650.755CharRedconjugateAlexaFluordyes[antibodyconjugates]MolecularProbes)Alexafluor350exafluor4054011028Alexafuor43054070AlexaFluor488643aY0.92Alexafluor500503525lexaFluor51451742714724Y0.61lexafluor5465721079aY0.79AlexaFluor5555531250QY01AlexaFluor568579792Alexafluor594591618820QY066Alexafluor610610Alexafluor6336481200AlexaFluor6476681300QY0.33Alexafluor6606636911100Alexafluor680807021150Alexafluor7006967191400Alexafluor7507521300Alexafluor7907828041750CyDvesCy2489506714QY0.12Cy3(512)5