Correlative light and electron microscopy (CLEM) combines light microscopy, for identifying a target via genetic labels, dyes, antibodies and morphological features, with electron microscopy, for analyzing high-resolution subcellular ultrastructures. Here, we describe step-by-step instructions to perform a CLEM experiment, optimized for the investigation of ultrastructural features in human brain tissue. The procedure is carried out at room temperature and can be adapted to other human and animal tissue samples. The procedure requires 8 d to complete and includes the stages of sample fixation for optimal ultrastructural preservation, immunofluorescence staining, image acquisition and multimodal image correlation and is executable within standard electron microscopy laboratories. Serving as a critical tool for characterizing human tissue and disease models, room-temperature CLEM facilitates the identification and quantification of subcellular morphological features across brain regions.