Hepcidin and ferroportin protein levels decreased in AD brain. a. Western blot analysis of the brain from frontal cortex demonstrated that a single 4 kDa band was clearly visible within all AD brain homogenates (lanes 4-6), in addition to a small proportion of control cortical tissue (lane 1-3). Ferroportin and hepcidin antibodies detected a single 62 kDa and 2.8 kDa band respectively in controls (lane 1-3), with less expression in AD. β-Actin loading control was used to normalize data. b. Densitometric analysis of the blots showed that there were significant differences between AD and control brains for all three peptides assessed. Aβ42 levels were higher in the diseased brain compared to controls (p<0.005) while hepcidin and ferroportin proteins were both significantly decreased (p<0.005 and p<0.01 respectively). c-n: Immunofluorescence in AD brains were assessed compared to healthy controls. Double staining was performed with polyclonal hepcidin and monoclonal ferroportin antibodies and counterstained with DAPI for nuclei. In the control brain superior frontal cortex hepcidin (c) and ferroportin (d) exhibited diffuse staining throughout the tissue. Both proteins co-localised in astrocytes (e). In AD brain, both proteins were visible along the parenchymal surface of blood vessels (f-g). Arrow indicates dual-labelling for hepcidin and ferroportin in small blood vessels (f-h). In the hippocampus of AD brains, hepcidin was visible in neurons with limited co-localisation with Aβ ( white arrow, i-l ). Ferroportin expression appeared to be reduced in AD brains (j). However, hepcidin was visible in damaged neurons (k). In control brain sections from mid temporal gyrus (MTG), hepcidin protein co-localised with GFAP (m). To evaluate specificity of primary antibodies, a mouse brain section was incubated with secondary antibodies and DAPI, with the omission of the primary antibody. There was no visible non-specific binding (n). Scale bar panels c-e and i-k &m 200 μm, f-h 100 μm, l = 25 μm.