Hepcidin

Paraffin-embedded carotid arteries were deparaffinized in xylene, rehydrated in graded ethanol (from 100% to 95% and then to 70%), and subjected to immunostaining. Immunohistochemistry was performed on serial sections, as previously described. The primary antibodies used were hepcidin (Santa Cruz Biotechnology Inc., Heidelberg, Germany), CD68 clone PG-M1 (DAKO), smooth muscle actin clone (SMA) 1A4 (DAKO), ferritin (DAKO), and transferrin receptor 1 (TfR1) (Alpha Diagnostic International, TX, USA). The immunoreactions were visualized using the EnVision+/HRP (DAKO) method and ChemMate EnVision Detection Kit (DAKO). Control sections without primary antibodies or with non-immune IgG were run for each protocol, resulting in consistently negative results. The slides were counterstained with hematoxylin.
All histological sections were examined under a light microscope, and the images were digitalized with the Image Grabber program (Toronto, ON, Canada). The microscope was set on the same parameters used to scan all samples. The randomly digitalized images were analyzed with Adobe Photoshop (v5.5), as described previously. The individual responsible for the analysis was blinded to patient information [13 (link)].