Immunohistochemical Analysis of Kidney Sections

Formalin-fixed, paraffin-embedded kidney sections were deparaffinized in xylene and rehydrated in ethanol containing distilled water. Tissue sections were stained using antibodies against C3c (1:10,000, A0062, Agilent Dako, Santa Clara, CA, USA) and C4d (1:50, 503-17344, Zytomed, Berlin, Germany), and labeling was performed using the Novolink^TM Polymer Detection System (Leica Biosystems, Wetzlar, Germany) according to the manufacturer’s protocol. Nuclear counterstaining was performed by using Mayer’s Hematoxylin Solution (Sigma, St. Louis, MO, USA). As previously described, kidney biopsies were evaluated for the presence/absence of C3c and C4d deposits in the glomerular tuft, interlobular arteries, peritubular capillaries, and venules [49 (link),50 (link)].