Rabbit monoclonal anti-Cthrc1 clone Vli-55 (www.mmcri.org/antibody ) was used for Western blotting and immunohistochemistry on paraffin-embedded, formalin-fixed tissues at 20 ng/ml following antigen retrieval with citrate buffer (0.1 M, pH = 6.0, 10 min in a steamer). This antibody had been previously characterized. It shows no immunoreactivity in any tissue of Cthrc1 null mice [14] (link). Subsequent steps of the immunostaining procedure were executed as previously published [14] (link). For immunostaining on tissues containing melanin-expressing cells a red color reaction product was chosen using a kit (VECTASTAIN ABC alkaline phosphatase, VECTOR Red Alkaline Phosphatase Substrate, Vector Laboratories). None of the mouse monoclonal antibodies were suitable for immunohistochemistry on paraffin sections. Immunoblotting was performed on cell lysates (CL) and conditioned media (CM) of HEK293T cells (293) and CHO-K1 cells (CHO) transduced with rat or human Cthrc1 expressing adenovirus as described [14] (link). Cthrc1 monoclonal antibodies were used at 10 ng/ml.
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