Vectastain abc alkaline phosphatase

Extracellular histone H3 levels were determined using a semi-quantitative method previously described^{50 (link),51 (link)}. Briefly, plasma dilutions were subjected to SDS-PAGE gel electrophoresis and transferred to PVDF membranes (Bio-Rad Laboratories, Hemel Hempstead, UK) using semi-dry blotting. Membranes were blocked and incubated with primary anti-H3 antibody, o/n at 4 °C, (sc-8654-R, Santa Cruz Biotechnology, Heidelberg, Germany), followed by a secondary biotin-conjugated IgG for 30 min at RT (ab97083, Abcam, Cambridge, UK) and a streptavidin–biotin/alkaline phosphatase complex (Vectastain ABC-Alkaline Phosphatase for 30 min at RT, Vector Laboratories, Burlingame, USA). Histone H3 bands were detected by luminescent ECL substrate (Advansta, San Jose, USA). Resulting band densities were quantified by ImageQuant TL software (GE Healtcare, Little Chalfont, UK), as compared to known concentrations of purified calf thymus H3 (Roche, Basel, Switzerland). This analysis is independent of frequently observed cross reactivity of histone antibodies with non-histone plasma proteins and allows the inspection of potential in vivo histone proteolytic processing.

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.