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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, 2496 views, 0 Q&A, How to cite
DOI: http://dx.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.

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.


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)
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