实验四细胞荧光测定术 (Cytofluorimetry) 17 核DNA的测定 (1)荧光锡夫型试剂(fluorescent schiff-type reagent) 副品红(pararosaniline) 金胺O (auramine O) 吖黄素 (acriflavine) 吖啶黄(acridine yellow) BAO (bis-[4-aminopheny]-1,3,4,-oxadiazole) Plant Reproduction Biology
1 Plant Reproduction Biology 实验四 细胞荧光测定术 (Cytofluorimetry) 1 核DNA的测定 ( 1 ) 荧光锡夫型试剂 ( fluorescent schiff-type reagent) 副品红(pararosaniline) 金胺O (auramine O) 吖黄素 (acriflavine) 吖啶黄(acridine yellow) BAO (bis-[4-aminopheny]-1,3,4,-oxadiazole)
1核DNA的测定 (2)锭和咪类 Hoechst 33258 DAPI (4,6-diamidino-2-phenylindole) 0.01~0.05%UV激发 EB(溴化乙锭,ethidium bromide) PI(碘化乙锭propidium iodide) 光辉酶素(mithramycin) (3)现代检测探针(见www.probes.com/mailinglist) 2 Plant Reproduction Biology
2 Plant Reproduction Biology 1 核DNA的测定 (2)锭和咪 类 Hoechst 33258 DAPI (4`,6-diamidino-2-phenylindole) 0.01~0.05% UV 激发 EB (溴化乙锭,ethidium bromide) PI (碘化乙锭propidium iodide) 光辉酶素(mithramycin) (3)现代检测探针(见www.probes.com/mailinglist)
实验四细胞荧光测定术 (Cytofluorimetry 2RNA染料 吖啶橙 3细胞壁染料 荧光增白剂(Calcafluor white)0.02%UV激发 4胼胝质染料 苯胺蓝(Aniline blue)0.002%UV激发 3 Plant Reproduction Biology
3 Plant Reproduction Biology 实验四 细胞荧光测定术 (Cytofluorimetry) 2 RNA染料 吖啶橙 3 细胞壁染料 荧光增白剂(Calcafluor white)0.02% UV 激发 4 胼胝质染料 苯胺蓝(Aniline blue) 0.002% UV 激发
实验四细胞荧光测定术 (Cytofluorimetry) 5细胞生活力测定 FDA(荧光素二醋酸酯,fluorescein diacewtate) 0.1%蓝光 激发 4 Plant Reproduction Biology
4 Plant Reproduction Biology 实验四 细胞荧光测定术 (Cytofluorimetry) 5 细胞生活力测定 FDA(荧光素二醋酸酯,fluorescein diacewtate) 0.1%蓝光 激发
实验四细胞荧光测定术 (Cytofluorimetry) 6细胞器的观察和定位 ●线粒体 JC-1 (5.5',6.6'-tetrachloro-1.1',3.3'-tetraethybenzimidaly Icarbocyanine iodide. 激光波长=514nm,发射波长529nm) 罗达明123(rhodamine123), 激光波长528nm,发射波长550nm) 5 Plant Reproduction Biology
5 Plant Reproduction Biology 实验四 细胞荧光测定术 (Cytofluorimetry) 6 细胞器的观察和定位 ●线粒体 JC-1(5.5’ , 6.6’-tetrachloro-1.1’ , 3.3’-tetraethybenzimidaly lcarbocyanine iodide, 激光波长=514nm, 发射波长529nm) 罗达明123(rhodamine 123), 激光波长528nm,发射波长550nm)
6细胞器的观察和定位 。溶酶体 DAMP[N-(3-1(2,4-dinitrophenyl)amino)propyl]-N- (3-aminopropyl)methylamine,dihydrochloride] 激发入=346nm 中性红(neutral red), 激光入=541nm,发射入=640nm 6 Plant Reproduction Biology
6 Plant Reproduction Biology 6 细胞器的观察和定位 ●溶酶体 DAMP[N-(3-1(2,4-dinitrophenyl)amino)propyl]-N- (3-aminopropyl)methylamine, dihydrochloride] 激发λ=346nm 中性红(neutral red), 激光λ=541nm, 发射λ=640nm
6细胞器的观察和定位 ● 内质网 DioC6 (3) (3,3'-dihexyloxacarbocyanineviodide) 激发λ=459nm,发射入=584nm 7 Plant Reproduction Biology
7 Plant Reproduction Biology 6 细胞器的观察和定位 ●内质网 DioC6(3) (3,3’-dihexyloxacarbocyanineviodide) 激发λ=459nm,发射λ=584nm
实验四细胞荧光测定术 (Cytofluorimetry ●高尔基复合体 NBD Ceramide (6-C (N-(7-nitrobenz-2- oxa-1,3-diazol-4-yl)anmino) (aproyl)sphingosine) 激发入=464nm,发射λ=532nm 8 Plant Reproduction Biology
8 Plant Reproduction Biology ●高尔基复合体 NBD Ceramide(6-C(N-(7-nitrobenz-2- oxa-1,3-diazol-4-yl)anmino) (aproyl)sphingosine) 激发λ=464nm,发射λ=532nm 实验四 细胞荧光测定术 (Cytofluorimetry)
实验四细胞荧光测定术 (Cytofluorimetry) 7.细胞骨架 ●微管骨架(microtubule)-间接免疫 荧光检测 ○a-微管蛋白→免血液→抗a-微白的单 克隆抗体(anti-a-tubulin,抗体1) 9 Plant Reproduction Biology
9 Plant Reproduction Biology 实验四 细胞荧光测定术 (Cytofluorimetry) 7.细胞骨架 ●微管骨架(microtubule)-间接免疫 荧光检测 ○α-微管蛋白→免血液→抗α-微白的单 克隆抗体(anti-α-tubulin,抗体1)
实验四细胞荧光测定术 (Cytofluorimetry 抗体1→老鼠血液→抗体2(anti-mouse IgG FITC Conjuagated) *FTC:异硫氰酸荧光素, 激发λ=490nm(兰光) 发射入=520nm(绿光) 10 Plant Reproduction Biology
10 Plant Reproduction Biology ○抗体1→老鼠血液→抗体2(anti-mouse IgG FITC Conjuagated) *FITC:异硫氰酸荧光素, 激发λ=490nm(兰光) 发射λ=520nm(绿光) 实验四 细胞荧光测定术 (Cytofluorimetry)