Immunostaining Protocol: P-Smad2 (Xenograft and Mice)

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-Smad2 (Cell Signaling Technology, catalog number: 3108 )
   
6. BrightVision poly-HRP anti- Rabbit (Immunologic, catalog number: DPVR-110HRP )
   
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. Citrate buffer (pH 6) (see Recipes)
    

Equipment

1. Oven
   
2. Immunostaining apparatus
   
3. Autoclave
   

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. Citrate Buffer (pH 6)
      
   2. Time: 20 min autoclave (121 °C)
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 1ml 1x wash buffer.
   
7. Incubation with primary antibody.
   1. Antibody: Rabbit anti-P-Smad2
      
   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 distilled water.
    
13. Hematoxilin (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-SMAD2 staining (arrowhead) in liver metastasis generated after intrasplenic injection of CRC cells. E: epithelial cells, Str: stromal cells. Scale bars = 10 μm

Recipes

1. Citrate buffer (pH 6)
   Citrate 5.8 g sodium citrate
   Set pH 6 with citric acid
   2 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.