Immunohistochemistry of wild type and hjv-/- iron manipulated mouse livers and spleens treated with LPS or Hepcidin|铁代谢数据集|炎症反应数据集

The iron hormone hepcidin is transcriptionally activated by iron or inflammation via distinct, partially overlapping pathways. We addressed how iron affects inflammatory hepcidin levels and the ensuing hypoferremic response. Dietary iron overload did not mitigate hepcidin induction in LPS-treated wt mice but prevented effective inflammatory hypoferremia. Likewise, LPS modestly decreased serum iron in hepcidin-deficient Hjv-/- mice, model of hemochromatosis. Synthetic hepcidin triggered hypoferremia in control but not iron-loaded wt animals. Furthermore, it dramatically decreased hepatic and splenic ferroportin in Hjv-/- mice on standard or iron-deficient diet, but only triggered hypoferremia in the latter. Mechanistically, iron antagonized hepcidin responsiveness by inactivating IRPs in the liver and spleen, to stimulate ferroportin mRNA translation. Prolonged LPS treatment eliminating ferroportin mRNA permitted hepcidin-mediated hypoferremia in iron-loaded mice. Thus, de novo ferroport..., Tissue specimens were fixed in 10% buffered formalin and embedded in paraffin. Samples from 3 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 de-paraffinization and heat-induced epitope retrieval. Immunostaining was performed by using 1:500 diluted rabbit polyclonal antibodies against ferroportin 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 four minutes, blued with Bluing Reagent for four minutes, 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 conventio..., Aperio ImageScope