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