Cytochrome-c release

Neurons treated with 5 μM Aβos in the presence or absence of Wnt3a for 24 h were loaded with MitoTracker Orange (50 nM), fixed and then analysed by immunofluorescence using a mouse anti-cytochrome-c antibody (BD Pharmingen, San Diego, CA) to detect cytochrome c localization. Hoechst staining was used to detect live cells and only those neurons with no condensed nucleus were considered for the analyses. Images were captured with an Olympus FluoView1000 Confocal Microscope and analyzed using NIH ImageJ software. Manders’ Coefficient M2 was calculated to determine the colocalization of cytochrome c with mitochondria.