Analyzing ACE Localization in Fibroblasts

WT fibroblasts, PS1-KO fibroblasts transfected with F-ACE, and PS1-KO fibroblasts transfected with F-ACE and PS1 mutants were seeded using image culture dishes (Eppendorf) and incubated at 37°C for 24 h. We firstly fixed cells in 4% paraformaldehyde for 30 min at room temperature. The cells were then permeabilized with 0.1% Triton X-100 for 20 min and incubated in 10% donkey serum in Tris-buffered saline containing 0.05% Tween 20 for 1 h at room temperature. The cells were incubated overnight at 4°C with anti-syntaxin-6 and anti-ACE antibodies. Immunofluorescent labeling by staining with Alexa Fluor 488 or Alexa Fluor 568-conjugated secondary antibodies. Images were acquired with a confocal microscope (Olympus FV3000, Tokyo, Japan). Details on the antibodies used are provided in Supplementary Table S1. ImageJ software was used to quantify the colocalization of ACE with the Golgi apparatus. The threshold intensity for both fluorescent signals is preset, which is determined using a colocalization threshold function. Colocalized pixels above a threshold intensity were automatically quantified and scored, and results were expressed as colocalized mean intensity positivity for both channels. Supplementary Table S1 provides details on antibodies and reagents.