Protocol to study synapse density or volume—SynDOVE—in brain using confocal microscopy and Imaris three-dimensional surface rendering software

We present a protocol for analyzing pre- and postsynaptic loci in fixed brain sections using confocal microscopy images and Imaris software. We describe steps for capturing z stack images of immunofluorescent-labeled synaptic proteins using a confocal microscope. We then detail procedures for deconvolving images, rendering three-dimensional (3D) surface recontructions of synaptic markers, and isolating closely juxtaposed pre- and post-synaptic 3D surfaces, herein termed “synaptic loci.” This protocol allows for quantitation of synaptic loci density, as well as pre- and postsynaptic volumes. For complete details on the use and execution of this protocol, please refer to Gcwensa et al. 1 • Guidance on navigating Imaris interface for synapse density and volume analysis • Protocol for rendering three-dimensional surfaces of synaptic proteins • Step-by-step guide to isolate synaptic loci and generate appropriate statistics • Procedure for calculating density of synaptic loci and volume of puncta Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. We present a protocol for analyzing pre- and postsynaptic loci in fixed brain sections using confocal microscopy images and Imaris software. We describe steps for capturing z stack images of immunofluorescent-labeled synaptic proteins using a confocal microscope. We then detail procedures for deconvolving images, rendering three-dimensional (3D) surface reconstructions of synaptic markers, and isolating closely juxtaposed pre- and post-synaptic 3D surfaces, herein termed “synaptic loci.” This protocol allows for quantitation of synaptic loci density, as well as pre- and post-synaptic volumes.