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We sought to understand the mechanisms behind the potent effect of stromal TGF-beta program on the capacity of colorectal cancer (CRC) cells to initiate metastasis. We discovered that mice subcutaneous tumors and metastases generated in the context of a TGF-beta activated microenvironment displayed prominent accumulation of p-STAT3 in CRC cells compared with those derived from control cells. STAT3 signaling depended on GP130 as shown by strong reduction of epithelial p STAT3 levels upon GP130 shRNA-mediated knockdown in CRC cells.

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Immunostaining Protocol: P-Stat3 (Xenograft and Mice)
免疫染色法:P-Smat3(磷酸化-Smad3,异种移植和小鼠)

癌症生物学 > 通用技术 > 生物化学试验 > 蛋白质分析
作者: Alexandre Calon
Alexandre CalonAffiliation: Oncology Programme, Institute for Research in Biomedicine, Barcelona, Spain
Bio-protocol author page: a1329
Elisa Espinet
Elisa EspinetAffiliation: Oncology Programme, Institute for Research in Biomedicine, Barcelona, Spain
Bio-protocol author page: a1330
Sergio Palomo-Ponce
Sergio Palomo-PonceAffiliation: Oncology Programme, Institute for Research in Biomedicine, Barcelona, Spain
Bio-protocol author page: a1331
Daniele V. F. Tauriello
Daniele V. F. TaurielloAffiliation: Oncology Programme, Institute for Research in Biomedicine, Barcelona, Spain
Bio-protocol author page: a1332
Mar Iglesias
Mar IglesiasAffiliation: Department of Pathology, Hospital Universitari del Mar, Barcelona, Spain
Bio-protocol author page: a1333
María Virtudes Céspedes
María Virtudes CéspedesAffiliation: IIB Sant Pau, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain
Bio-protocol author page: a1334
Marta Sevillano
Marta SevillanoAffiliation: Oncology Programme, Institute for Research in Biomedicine, Barcelona, Spain
Bio-protocol author page: a1335
Cristina Nadal
Cristina NadalAffiliation: Institut de Malalties Hemato-Oncològiques, Hospital Clínic, Barcelona, Spain
Bio-protocol author page: a1336
Peter Jung
Peter JungAffiliation: Oncology Programme, Institute for Research in Biomedicine, Barcelona, Spain
Bio-protocol author page: a1337
Xiang H.-F. Zhang
Xiang H.-F. ZhangAffiliation: Cancer Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, New York, USA
Bio-protocol author page: a1338
Daniel Byrom
Daniel ByromAffiliation: Chemistry and Molecular Pharmacology Programme, Institute for Research in Biomedicine, Barcelona, Spain
Bio-protocol author page: a1339
Antoni Riera
Antoni RieraAffiliation: Chemistry and Molecular Pharmacology Programme, Institute for Research in Biomedicine, Barcelona, Spain
Bio-protocol author page: a1340
David Rossell
David RossellAffiliation: Biostatistics and Bioinformatics Unit, Institute for Research in Biomedicine, Barcelona, Spain
Bio-protocol author page: a1341
Ramón Mangues
Ramón ManguesAffiliation: IIB Sant Pau, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain
Bio-protocol author page: a1342
Joan Massague
Joan MassagueAffiliation: Cancer Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, New York, USA
Bio-protocol author page: a1343
Elena Sancho
Elena SanchoAffiliation: Oncology Programme, Institute for Research in Biomedicine, Barcelona, Spain
For correspondence: elena.sancho@irbbarcelona.org
Bio-protocol author page: a1344
 and Eduard Batlle
Eduard BatlleAffiliation: Oncology Programme, Institute for Research in Biomedicine, Barcelona, Spain
For correspondence: eduard.batlle@irbbarcelona.org
Bio-protocol author page: a1345
Vol 4, Iss 9, 5/5/2014, 3285 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1120

[Abstract] We sought to understand the mechanisms behind the potent effect of stromal TGF-beta program on the capacity of colorectal cancer (CRC) cells to initiate metastasis. We discovered that mice subcutaneous tumors and metastases generated in the context of a TGF-beta activated microenvironment displayed prominent accumulation of p-STAT3 in CRC cells compared with those derived from control cells. STAT3 signaling depended on GP130 as shown by strong reduction of epithelial p STAT3 levels upon GP130 shRNA-mediated knockdown in CRC cells.

[Abstract] 我们试图了解基质TGF-β程序对结肠直肠癌(CRC)细胞启动转移能力的有效作用背后的机制。 我们发现小鼠皮下肿瘤和转移生成的TGF-β激活微环境的情况下显示显着积累的p-STAT3在CRC细胞相比那些衍生自控制细胞。 STAT3信号传导依赖于GP130,如通过GP130shRNA介导的CRC细胞敲除的上皮p STAT3水平的强烈减少所示。

Materials and Reagents

  1. Paraffin sections (subcutaneous tumors samples or liver metastasis from nude mice respectively injected subcutaneously or intrasplenic with CRC cells)
  2. XILOL
    Note: Xylol also referred to as xylene or dimethylbenzene is a solvent used in histology as a clearing agent to remove paraffin from dried microscope slides prior to staining.
  3. MilliQ H2O
  4. Wash buffer (Dako, catalog number: K800721 )
  5. Rabbit anti-P-Stat3 (Cell Signaling Technology, catalog number: 9145S )
  6. BrightVision poly-HRP anti- Rabbit (Immunologic, catalog number: DPVR110HRP )
  7. Envision FLEX antibody diluent (Dako, catalog number: K8006 )
  8. Peroxidase Blocking Solution (Dako, catalog number: S202386 )
  9. ImmPACT DAB (Vector Laboratories, catalog number: SK-4105 )
  10. DPX mounting media (Sigma-Aldrich, catalog number: 06522 )
  11. Hematoxylin
  12. Tris/EDTA (pH 9.0) (see Recipes)

Equipment

  1. Oven
  2. Immunostaining apparatus

Procedure

  1. Stove samples at 65 °C just before starting the immunostaining technique. Remove the samples from the oven when the wax present in sections is completely undone.
  2. De-waxing and rehydration: Place slides in a rack to perform following washes (bath).
    1. XILOL: 10 min
    2. XILOL: 10 min
    3. XILOL: 5 min
    4. 100% EtOH: 10 min
    5. 100% EtOH: 5 min
    6. 96% EtOH: 5 min
    7. 90% EtOH: 10-15 times
    8. 80% EtOH: 10-15 times
    9. 70% EtOH: 10-15 times
    10. 50% EtOH: 10-15 times
    11. 25% EtOH: 10-15 times
    12. H2O MilliQ: 10-15 times
  3. Antigen retrieval.
    1. Tris-EDTA Buffer (pH 9.0)
    2. Time: 20 min in boiling water
  4. 3 washes 5 min with 1 ml 1x wash buffer.
  5. Blocking endogenous peroxidase.
    1. 200 ml Peroxidase Blocking Solution
    2. Time: 10 min
  6. 3 washes 5 min with 1 ml 1x wash buffer.
  7. Incubation with primary antibody.
    1. Antibody: Rabbit anti-P-Stat3
    2. Dilution 1/200 in Envision FLEX antibody diluent
    3. 200 µl/sample
    4. O/N 4 °C
  8. 3 washes 5 min with 1 ml 1x wash buffer
  9. Incubation with antibody BrightVision
    1. Antiboby: BrightVision anti-Rabbit
    2. 150 µl/sample
    3. Time: 45 min at room temperature
  10. 3 washes 5 min with 1 ml 1x wash buffer
  11. Revealed with ImmPACT DAB.
    1. 200 µl/sample
    2. Time: 10 min
  12. 3 washes 5 min with 1 ml distilled water.
  13. Hematoxylin (1 ml) counterstaining, time: 2 min.
  14. Rinse in distilled water bath.
  15. Rinse in TAP water bath.
  16. Dehydration and mounting with DPX.

Representative data



Figure 1. p-STAT3 staining of liver metastases generated after intrasplenic injection of CRC cells. Note strong staining in epithelial cells (arrows). E: epithelial cells, Str: stromal cells. Scale bar = 50 µm

Recipes

  1. Tris/EDTA (pH 9.0)
    12 g Tris
    3.7 g EDTA in 10 L MilliQ

Acknowledgments

A.C. and D.V.F.T hold a Juan de la Cierva postdoctoral fellowship, E.E. an FPI PhD fellowship (both from Spanish Ministry of Science and Innovation). This work has been supported by grants from Instituto de Salud Carlos III FEDER (RD09/0076/00036) and the “Xarxa de Bancs de tumors sponsored by Pla Director d’Oncologia de Catalunya (XBTC), to E.B. from the European Research Council (Starting grant - 208488) and Consolider programmes (MICINN), to E.S. and A.C. by the Spanish Ministry of Science and Innovation, to JM by NIH grant CA34610 and to RM by grants PS09/00965 (MICINN) and NanoCoMets (CIBERBBN).

References

  1. Calon, A., Espinet, E., Palomo-Ponce, S., Tauriello, D. V., Iglesias, M., Cespedes, M. V., Sevillano, M., Nadal, C., Jung, P., Zhang, X. H., Byrom, D., Riera, A., Rossell, D., Mangues, R., Massague, J., Sancho, E. and Batlle, E. (2012). Dependency of colorectal cancer on a TGF-beta-driven program in stromal cells for metastasis initiation. Cancer Cell 22(5): 571-584.

材料和试剂

  1. 石蜡切片(皮下肿瘤样品或分别从皮下或脾内注射CRC细胞的裸鼠的肝转移)
  2. XILOL
    注意:二甲苯也称为二甲苯或二甲苯是在组织学中用作清除剂的溶剂,以在染色前从干燥的显微镜载玻片上除去石蜡。
  3. MilliQ H sub 2 O 2 /
  4. 洗涤缓冲液(Dako,目录号:K800721)
  5. 兔抗P-Stat3(Cell Signaling Technology,目录号:9145S)
  6. BrightVision poly-HRP抗兔(Immunologic,目录号:DPVR110HRP)
  7. Envision FLEX抗体稀释剂(Dako,目录号:K8006)
  8. 过氧化物酶封闭溶液(Dako,目录号:S202386)
  9. ImmPACT DAB(Vector Laboratories,目录号:SK-4105)
  10. DPX安装介质(Sigma-Aldrich,目录号:06522)
  11. 苏木精
  12. Tris/EDTA(pH 9.0)(参见配方)

设备

  1. 烤箱
  2. 免疫染色仪

程序

  1. 在开始免疫染色技术之前在65℃下烘烤样品。 当存在于切片中的蜡完全去除时,从烘箱中取出样品
  2. 脱蜡和再水合:将玻片放在架子上进行以下洗涤(浴)。
    1. XILOL:10分钟
    2. XILOL:10分钟
    3. XILOL:5分钟
    4. 100%EtOH:10分钟
    5. 100%EtOH:5分钟
    6. 96%EtOH:5min
    7. 90%EtOH:10-15倍
    8. 80%EtOH:10-15倍
    9. 70%EtOH:10-15倍
    10. 50%EtOH:10-15倍
    11. 25%EtOH:10-15倍
    12. H 2 O MilliQ:10-15次
  3. 抗原检索。
    1. Tris-EDTA缓冲液(pH9.0)
    2. 时间:在沸水中20分钟
  4. 3次用1ml 1x洗涤缓冲液洗涤5分钟
  5. 阻断内源性过氧化物酶。
    1. 200 ml过氧化物酶封闭溶液
    2. 时间:10分钟
  6. 3用1ml 1x洗涤缓冲液洗涤5分钟。
  7. 与第一抗体孵育。
    1. 抗体:兔抗P-Stat3
    2. 稀释1/200在Envision FLEX抗体稀释剂
    3. 200μl/样品
    4. O/N 4℃
  8. 3用1ml 1x洗涤缓冲液洗涤5分钟
  9. 与抗体BrightVision孵育
    1. Antiboby:BrightVision抗兔
    2. 150μl/样品
    3. 时间:室温下45分钟
  10. 3用1ml 1x洗涤缓冲液洗涤5分钟
  11. 揭示了ImmPACT DAB。
    1. 200μl/样品
    2. 时间:10分钟
  12. 3用1ml蒸馏水洗涤5分钟
  13. 苏木精(1ml)复染色,时间:2分钟。
  14. 用蒸馏水冲洗。
  15. 在TAP水浴中冲洗。
  16. 用DPX脱水和安装。

代表数据



图1.脾细胞注射CRC细胞后产生的肝转移瘤的p-STAT3染色。注意上皮细胞中强染色(箭头)。 E:上皮细胞,Str:基质细胞。 比例尺=50μm

食谱

  1. Tris/EDTA(pH9.0)
    12克Tris
    3.7g EDTA在10L MilliQ中

致谢

A.C.和D.V.F.T持有Juan de la Cierva博士后研究金,E.E. FPI博士研究生奖学金(均来自西班牙科学和创新部)。这项工作得到了Salud Carlos III FEDER研究所(RD09/0076/00036)和"加拿大肿瘤学院院长赞助的肿瘤研究"(XBTC)给予E.B.的资助。从欧洲研究委员会(启动补助金 - 208488)和Consolider计划(MICINN)到E.S.由西班牙科学和创新部授予JM,NIH授予JM,CA34610授予JM,通过授予PS09/00965(MICINN)和NanoCoMets(CIBERBBN)授予RM。

参考文献

  1. Calon,A.,Espinet,E.,Palomo-Ponce,S.,Tauriello,DV,Iglesias,M.,Cespedes,MV,Sevillano,M.,Nadal,C.,Jung,P.,Zhang,XH,Byrom D.,Riera,A.,Rossell,D.,Mangues,R.,Massague,J.,Sancho,E。和Batlle,E。(2012)。 结肠直肠癌对基质细胞中TGF-β驱动的程序对转移起始的依赖性。 a> Cancer Cell 22(5):571-584。
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How to cite this protocol: Calon, A., Espinet, E., Palomo-Ponce, S., Tauriello, D. V., Iglesias, M., Céspedes, M. V., Sevillano, M., Nadal, C., Jung, P., Zhang, X. H., Byrom, D., Riera, A., Rossell, D., Mangues, R., Massague, J., Sancho, E. and Batlle, E. (2014). Immunostaining Protocol: P-Stat3 (Xenograft and Mice). Bio-protocol 4(9): e1120. DOI: 10.21769/BioProtoc.1120; Full Text



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Alexandre Calon的其他实验方案(1)
Elisa Espinet的其他实验方案(1)
Sergio Palomo-Ponce的其他实验方案(1)
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