原位杂交操作流程【各行参照】.ppt

原位杂交操作流程原位杂交操作流程1行业专项冰冻切片的（冰冻切片的（DIG-labeled RNA probe）原位杂交操作程序）原位杂交操作程序一、一、             切片制备切片制备用新鲜组织制作用新鲜组织制作6μm厚冰冻切片，厚冰冻切片，37℃干燥干燥1～～4小时，进行下一步操作小时，进行下一步操作二、二、             预杂交前处理预杂交前处理预固定预固定：：4%PFA（（in DEPC-PBS Ph7.4））RT 30～～60min    4%PFA：：多聚甲醛多聚甲醛     20g                                    1×PBS            450ml                                加热（加热（60℃、、20min）溶解）溶解                                  冷却后调冷却后调pH至至7.4                                    1×PBS    →      500mlPBS  RT  5 min×2                                                          10×PBS：：500ml                                                                                                        NaCl                 40g                                                                                                        KCl                     1g                                                                                                        KH2PO4            1g                                                                                                        Na2HPO4        7.7g                                                                                                        DEPC水水          450ml                                                                                           调调Ph至至7.5 DEPC水加至水加至500mlPBS(含含0.3%v/vTritonX-100) RT 15 min                  临用前配制临用前配制                                                                                                         10%TritonX-100    15ml                                                                                                         1×PBS    →     500ml    PBS   RT   5min ×22行业专项消化消化：： 2μg/mlProteinase K in TE buffer 37℃ 10 min          TE：：                                                                                                        pH8.0 Tris-HCl 1M  5ml                                                                                                       pH8.0 EDTA  0.5M   1ml                                                                                                            DEPC水水    →     500ml   0.2%Glycin in PBS   RT 5 min×2                                                                                                                              (50xDenhart’s;                                                                                                         Ficoll 400   聚蔗糖聚蔗糖  1g  后固定后固定：：4%PFA in PBS RT 15 min                                           PVP 聚乙烯基吡咯烷酮聚乙烯基吡咯烷酮 1g                                                                                                         BSA   牛血清白蛋白牛血清白蛋白   1g DEPC    水水       100ml））      PBS  RT 3 min×2 0.1 M  三乙醇胺（三乙醇胺（TEA））/0.25%乙酸酐乙酸酐 RT 5 min×2                                                                                              0.1M 三乙醇胺三乙醇胺：三乙醇胺：三乙醇胺 2.64ml                             DEPC水水   200ml                                                                                                            用前加入乙酸酐用前加入乙酸酐  500μlPBS  RT 5 min×23行业专项三、三、             预杂交预杂交 预杂交预杂交   50℃  2h                            预杂交液：预杂交液： 50%去离子甲酰胺去离子甲酰胺                                                                                      5×SSC                                                                                      5×Denhardt’s                                                                                       0.02%SDS                                                                                        0.1mg/mltRNA四、四、            杂交杂交杂交杂交   50℃  12h以上以上4行业专项 五、五、             杂交后处理杂交后处理2×SSC脱去盖膜（片）脱去盖膜（片）  50℃2×SSC清洗清洗  37℃  10 min×2         20μg/mlRNaseA in Rnsae buffer 37℃ 30 min         Rnsae buffer;2M Tris  5ml                                                                                                                     0.25MEDTA   4ml                                                                                                                     3MnaCl    167ml                                                                                                                       pH8.0                                                                                                                       D W→  1000ml    Rnase buffer 37℃ 30 min                                  2×SSC 50℃ 15 min×2                                     1×SSC（含（含0.02%SDS））37℃ 15 min×2                                            5%SDS          2ml                                                                                                                      1×SSC       500ml   0.1×SSC  37℃ 15 min×2                          5行业专项BufferⅠ 37℃ 10 min×2                          BufferⅠ:                                                                                   2M Tris-HCl Ph7.6  25ml                                                                                    3M  NaCl           25ml                                                                                     D.W.  →            500ml封闭；封闭；0.5%抗体阻断液抗体阻断液 in BufferⅠ（含（含0.2%Tween20））                                      37℃ 20 min                                         抗体阻断液抗体阻断液 :  (1:500抗地高辛抗体抗地高辛抗体 in 阻断液阻断液 37℃ 2 h)                         阻断剂阻断剂               1g                                                                                               Tween-20         0.4ml  BufferⅠ 37℃ 10 min×2                                                   BufferⅠ         200ml   BufferⅡ 37℃ 10 min×26行业专项 显显色色；； BufferⅡ:   2M Tris        10ml（（1:50NBT/BCIP Stock Solution in BufferⅡ 2~24h））             3M NaCl      6.67ml                                                                                                          MgCl2     2.033g（湿合、避光）（湿合、避光）                                                                              D.W.       180ml 浓浓HCl调调pH至至9.5                                                                                                          D.W.  →   200ml终止反应：终止反应： Buffer ⅢⅢ  RT  5min××2   流水冲洗流水冲洗 5-10min 1%甲基绿复染甲基绿复染3-10min，， 水洗，晾干，封固水洗，晾干，封固7行业专项DNA探针检测石蜡切片中DNA8行业专项组织标本•肝穿刺组织，常规石蜡包埋，4m连续石蜡切片，贴在涂有切片粘合剂的干净载玻片上，58℃烤18h。

9行业专项试剂试剂1 1．．2020××SSCSSC2 2．．HBV cDNA-DigHBV cDNA-Dig探探针 5 5 g g3 3．．4 4％多聚甲％多聚甲醛4 4．．0.1mol/L PBS0.1mol/L PBS，，pH7.4pH7.45 5．．0.2mmol/L HCl 0.2mmol/L HCl 和和0.1mol/L0.1mol/L甘氨酸甘氨酸 PBS pH7.4 PBS pH7.46 6．．0.4 0.4 ％％Tritor X-100 PBS pH7.4Tritor X-100 PBS pH7.47 7．蛋白．蛋白酶  20 20 g/mlg/ml8 8．．0.250.25％乙酸％乙酸酐，用，用0.1mol/L0.1mol/L三乙醇胺配制三乙醇胺配制9 9．．预杂交交缓冲液和冲液和杂交交缓冲液冲液10. AKP10. AKP标记抗抗Dig FabDig Fab段二抗，可段二抗，可1:5001:500稀稀释11. TSM11. TSM、、TSM2TSM2（配制参（配制参阅附附录4 4））12. NBT12. NBT和和BCIPBCIP显色液或用色液或用AKPAKP显色色KitKit 10行业专项操作流程•杂交前处理•预杂交•杂交•杂交后漂洗•杂交后检测•结果判断11行业专项杂交前处理1. 石蜡切片常规脱蜡至水2. 0.02M pH7.4 PBS洗3×3min3. 0.2M HCI 20min 室温 （除去蛋白）4. 2×SSC（内含5M EDTA） 50℃ 洗 30min5. 蛋白酶 2g/ml 37℃ 20min6. 0.1M甘氨酸PBS洗 10min 室温，中止酶反应7. 4％多聚甲醛，20min 室温8. PBS洗 3×3min9. 75％，85％，95％和无水乙醇各2min12行业专项预杂交和杂交         加预杂交液 20l/每片，42℃ 2h。

加杂交液10～20ul/每片（内含HBV-cDNA Dig标记探针4g/ml），加盖硅化玻片，将切片置于95℃ 10min，使探针和靶病毒DNA双链打开（变性），然后迅速置于冰上5min，再将切片置于盛有2×SSC湿合内，42℃杂交过夜(12～16h)13行业专项杂交后漂洗杂交后漂洗1. 将切片置于2×SSC液内振动移去盖片2. 2×SSC洗 2×10min， 55℃3. 0.5×SSC 2×5min， 50℃4. PBS洗3×3min5. 10％醋酸 20min 室温，阻断内源性碱性磷酸酶6. PBS洗3×3min14行业专项信号放大和显示信号放大和显示1. 1:500稀释AKP标记抗Dig二抗，37℃ 4h，或4℃过夜2. PBS洗 3×3min3. TSM1洗 2×5min4. TSM2洗 2×5min5.显色液 在1mlTSM2中加入4.5lNBT，3.5l， BCIP 混合后，显色30min～12h，中间不断观察6. TSM2洗，PBS洗3×3min，核固红或甲基绿衬染7. 蒸镏水洗，系列乙醇脱水，二甲苯透明，树脂封片15行业专项结果判断结果判断16行业专项ISHISH流程流程•探探针合成合成•组织细胞胞标本的准本的准备•ISHISH试剂的配制的配制•操作流程操作流程17行业专项探探   针针 1、根据文献报道合成PCR引物一对。

2、PCR扩增：用高保真DNA聚合酶 进行PCR扩增18行业专项 3、将PCR扩增产物亚克隆入pGEM2Z质粒（EcoRI和ACC I酶切位点），在大肠杆菌中扩增，纯化原理：PGEM3质粒购于promega公司，含有位于pUc19 MCS侧面的SP6和T7 RNA聚合酶启动子，在将所需序列插入MCS后，一个简单的基于lac Z α-肽灭活的颜色选择方案将促进重组体的筛选在体外，将SP6或T7 RNA聚合酶加入到含有标记物的三磷酸核苷前体的转录系统中，可允许从任何一方向产生多拷贝DNA插入序列的标记RNA探针19行业专项•4、  利用Roche生产的体外转录标记RNA kit（Cat.No 999644）操作流程参阅《原位检测技术》p132-13320行业专项•     组织细胞胞标本的准本的准备• ISHISH试剂的的配配制制（（所所有有试剂和和用用具具均用均用DEPCDEPC水水处理）理）21行业专项操作流程操作流程1  1、活细胞经4％多聚甲醛固定20min，室温，PBS洗，系列乙醇脱水2、组织标本：标准取材后，4％多聚甲醛固定6h，用25％遮糖或液氮速冻，切片贴在涂有APES胶的干净载玻片上。

3、PBS洗3×3min，冰冻切用4%柠檬酸盐90℃保温20min，石蜡切片98℃ 10min，细胞片不用4、2μg/ml蛋白酶K 37℃ 20min，PBS洗3×3min5、0.1mol/L甘氨酸PBS洗10min.6、0.25%乙酸酐的0.1mol/L三乙醇胺(pH8.0)洗10min，PBS洗10min22行业专项7 7、、预杂交交 0.2 0.2××SSCSSC＋＋5050％甲％甲酰胺胺 30min 37℃ 30min 37℃8 8、、杂交：滴加交：滴加杂交液（交液（2μg/ml2μg/ml）） 加一加一层复盖膜，复盖膜，48℃48℃杂交交8-12h8-12h9 9、、杂交后洗：交后洗： 2 2××SSCSSC洗洗 2 2××5min 45℃5min 45℃ 1 1××SSCSSC洗洗 2 2××5min 5min 室温室温 0.5 0.5××SSCSSC洗洗 2 2××5min 5min 室温室温 0.1 0.1××SSCSSC洗洗 2 2××5min 5min 室温室温 PBS PBS洗洗 3 3××3min3min23行业专项10、用10％正常血清封闭 30min11、抗Dig IgG 1:500 37℃ 1h12、PBS洗 3×3min13、0.02M TBS pH9.015min14、NBT/BCIP显色 1h-24h15、洗后，核固红衬染，蒸馏水洗，脱 水，透明，封片。

16、结果观察：紫蓝色为阳性信号，红 色为背景24行业专项。