第六讲免疫荧光组化技术

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第六讲免疫荧光组化技术. 主要内容一、荧光的特征二、荧光素三、荧光素标记抗体的方法四、荧光抗体的质量鉴定五、免疫荧光组化染色方法六、荧光显微镜七、非特异性荧光的消除方法八、激光扫描共聚焦显微镜. 思考题： 1. 什么叫荧光？ 2. 常用荧光素有哪些特征？ 3. 标记荧光抗体有哪二种主要的制备方法？. 4. 免疫荧光组化直接法、间接法的原理和主要操作流程？ 5. 观察荧光组化片时应注意哪些问题？ 6. 非特异性荧光的消除方法有哪几种？. - PowerPoint PPT Presentation

Transcript of 第六讲免疫荧光组化技术

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2.(1)FITC390D490495nm520530nm389.4
(2)TRITC550nm620nm
(3)(RB200) 570nm596600nm580RB200
(4)1--5-(5)4--4--2-SITS(6)Texas red(7)PE(8)Cyanine,Cy2,Cy3,Cy5
()FITC 1.Marsshall(1)FITC -FITCIgG861520
FITC-NC N- S H H FITC- N C N - H S H
(2) 50-100:120mg/ml 1/10 0.01mgFITC/1mg
SephadeG50 G25 4 -402.Chadwick3.4.
1.2.3.4.F/P
1. (1)PBS30minPBS
(2)371hPBS33min(3)0.01%13minPBS33min2Nacl(4)pH9.0(5)
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(1)PBSPBSPBS33min(2)371h4h4PBS33min
(3)37 30minPBS33min(4)0.01%13minPBS33min(5)
1. ()
(1) 515min5070
(2)
UDM400BP330-385BP360-370BA420DAPI()DNA Hoechest 33358,33342VDM455BP400-410BA455BVDM455BP400-440BA475BP420-440SBDM500BP450-480BA515BP470-490DNARNABP420-480IBDM505PB460-490BA515IFBP470-490GDM570BP510-550BA590BP530-550BP480-550DNAIGDM565BP520-550BA580IFDM600BP545-580BA610IF
2.(1)0.61.2mm(2)0.17mm(3)
(4) pH8.59.50.5mol/L pH9.09.5
mowiol 40-88 4.8g 12ml 12ml 0.2mol/L Tris(pH8.5) 24ml
5000g15min1.4-diazobicydo-[2,2,2]-octane(DABcos)2.5%(1.25g)-20(5)
3.(1)(2)(3)(4)12h90min
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1. 2. 3.Sephadex G-50 4.DEAE 5. 6. 7. 8.0.01%
(laser scanning confocal microscope,LSCM)80
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4. Confocal,pinhole,detector, PMT,Stepsize, X points, Y Points, scanstr,speed,LaserPwr,Auto zoom, Display, BR subval,Samples/pt
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1 ----1.4 m5818h2.10min9585752min3.PBS0.01mol/L pH7.433min
4.0.1%100ml100mg100mg0.1N NaoHpH7.63730min5.0.01mol/L pH7.4 PBS33min6.AFP1:50371h4h4
7.PBS33min8.IgG-F1:20371h2h9. PBS33min10.3min11.pH9.012.