Quantitative Fluorescent Imaging of Synaptic Proteins

Image analysis was carried out using a Zeiss Axio Imager A2 fluorescent microscope (Zeiss, Jena, Germany) with Cool Snap EZ camera (Visitron System, Puchheim, Germany) and MetaMorph Imaging software (MDS Analytical Technologies, Ismaning, Germany). Up to 3 coverslips were treated individually and processed per group. For each coverslip, the same exposure time and intensity were taken among the different groups. After background subtraction, the fluorescence intensity of the immunosignal was measured along dendrites right after the first branching point using ImageJ software. The synaptic immunofluorescence intensities of pan-AKT and phospho-AKT were assessed in a region of 400 nm × 400 nm square set by the mask generated based on synaptic marker Shank3. The Shank3 mask was created semi-automatically using OpenView software [34 (link)].