Confocal Microscopy Imaging and Quantification of Synaptic Immunofluorescence

Images were taken with a ZEISS LSM 880 Airyscan confocal laser scanning microscope equipped with ZEN2.1 software, using a Plan-Apochromat 63×/1.4 Oil DIC M27 63× oil objective. Preparation of images and quantification of the immunofluorescence (IF) was done using Image J (National Institutes of Health, NIH, http://rsb.info.nih.gov/ij/) and OpenView software [57 (link)]. Firstly, the background was subtracted from all channels using Image J, and the synaptic IF intensities were assessed in a region of interest (along 20 µm of the proximal dendrite) which was set by the mask in the channel for synaptophysin. This mask was created semi-automatically by using OpenView. The final adjustment of images was made by ImageJ and Photoshop (Adobe Systems, San Jose, CA, USA).