Immunohistochemistry of Ferroportin in Mice

Tissue specimens were fixed in 10% buffered formalin and embedded in paraffin. Samples from three different mice for each experimental condition were cut at 4 µm, placed on SuperFrost/Plus slides (Fisher) and dried overnight at 37°C. The slides were then loaded onto the Discovery XT Autostainer (Ventana Medical System) for automated immunohistochemistry. Slides underwent deparaffinization and heat-induced epitope retrieval. Immunostaining was performed by using 1:500 diluted rabbit polyclonal antibodies against ferroportin (Maffettone et al., 2010 (link)) and an appropriate detection kit (Omnimap rabbit polyclonal HRP, #760–4311 and ChromoMap-DAB #760–159; Roche). Negative controls were performed by the omission of the primary antibody. Slides were counterstained with hematoxylin for 4 min, blued with Bluing Reagent for 4 min, removed from the autostainer, washed in warm soapy water, dehydrated through graded alcohols, cleared in xylene, and mounted with Permount (Fisher). Sections were analyzed by conventional light microscopy and quantified by using the Aperio ImageScope software (Leica Biosystems; Fillebeen et al., 2018 (link)).