Development of a rabbit monoclonal antibody group against Smads and immunocytochemical study of human and mouse embryonic stem cells.

We report here the development of a group of rabbit monoclonal antibodies against Smad1, Smad2, Smad3, and Smad5, and the immunocytochemistry (ICC) staining of human embryonic stem cells (hESC) and mouse embryonic stem cells (mESC). Eight New Zealand rabbits were immunized with synthesized peptides linked to KLH, and splenocytes from these rabbits were fused with rabbit immortal B cell 240E-W2. Resulting hybridomas producing anti-Smad monoclonal antibodies were screened by enzyme-linked immunosorbent assay (ELISA) with BSA-linked peptides. Clones were chosen for antibody production based on their activities in Western blotting and on paraffin-embedded human tissues, and the capacity of the antibodies in immunocytochemistry was demonstrated. Using these antibodies, we performed ICC staining on routinely cultured human and mouse embryonic stem cells, and showed that both cell types strongly express these genes. We propose that both hESCs and mESCs have the ability to transduce signals from both BMPs and TGF-b/Activin.