Protocol for fixation and staining of postmortem human brain tissue optimized for focused ion beam-scanning electron microscopy

Here, we present a protocol for generating postmortem human brain tissue samples suitable for volume electron microscopy (vEM) imaging via focused ion beam-scanning electron microscopy (FIB-SEM). We describe steps for immersion fixation, sectioning, long-term cryopreservation, and enhanced-contrast staining that produce samples suitable for high-resolution FIB-SEM. This approach generates ultrastructure of sufficiently high preservation to enable both dense and targeted three-dimensional (3D) reconstructions of cellular, subcellular, synaptic, and organellar features in postmortem human brain samples. For complete details on the use and execution of this protocol, please refer to Glausier et al. 1 • Protocol to prepare postmortem human brain tissue for vEM imaging via FIB-SEM • Instructions for brain tissue fixation and long-term cryopreservation • Procedures to achieve uniform, high-contrast heavy metal staining • Guidance on indefinitely preserving processed tissue samples in Epon resin Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Here, we present a protocol for generating postmortem human brain tissue samples suitable for volume electron microscopy (vEM) imaging via focused ion beam-scanning electron microscopy (FIB-SEM). We describe steps for immersion fixation, sectioning, long-term cryopreservation, and enhanced-contrast staining that produce samples suitable for high-resolution FIB-SEM. This approach generates ultrastructure of sufficiently high preservation to enable both dense and targeted three-dimensional (3D) reconstructions of cellular, subcellular, synaptic, and organellar features in postmortem human brain samples.