Sc 20137

Immunohistochemical techniques were performed, according to standard protocols. Briefly, frozen tissue sections were fixed, cryostat sectioned and stained with a rabbit anti-human C1q antibody (A0136; Dako), a goat anti-human C3 antibody (sc-20137; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), a rabbit anti-human CR2 (HPA052942, Sigma-Aldrich, St. Lous, MO, USA) or with respective isotype control antibodies, and then washed and incubated with HRP-conjugated anti-rabbit or anti-goat IgG secondary Abs. Afterwards, tissue slides were incubated with DAB substrate (Dako) and counterstained with Mayer`s hemalum solution.

Whole-protein extracts were prepared by lysing biopsy or fecal samples in denaturing lysis buffer containing 1% SDS, 10 mM Tris (pH 7.4), and 1% protease inhibitor mixture (Complete Protease Inhibitor Cocktail; Roche Applied Science, Mannheim, Germany). Forty micrograms of protein extracts were separated by denaturing SDS-PAGE under reducing conditions and transferred onto polyvinylidene difluoride membranes. After blocking, the membranes were probed with C3-specific primary Ab (sc-20137, Santa Cruz Biotechnology, LLC, Solon, OH, USA) or a human IgM-specific primary Ab (A80-100A, Biomol, Hamburg, Germany), washed, and incubated with HRP-conjugated IgG as secondary Ab. The human IgA or IgG level was detected using HRP-conjugated IgG directed either against the human alpha chain (PA1-74395, Thermo Fisher Scientific) or against the human gamma chain (62-8420, Thermo Fisher Scientific). The proteins were visualized by chemiluminescence. To determine similar transfer and equal loading, the membranes were stripped and reprobed with an Ab specific for β-Actin (Sigma-Aldrich, St. Louis, MO, USA).