Expansion microscopy (ExM) is a new approach to super-resolution that relies on the physical isotropic expansion of a sample in a swellable polymer network. We developed a novel ExM method termed Sevenfold Post-labelling Expansion Microscopy (SP-ExM) that enables sevenfold expansion in a single step of thick cerebellar mouse brain and human tumour and peritumoral cortex samples, while preserving proteins for bulk staining of the entire proteome. Using SP-ExM, we localized proteins with nanoscale precision in their ultrastructural context without any specialized procedures or equipment. As such, SP-ExM contributes to better understanding the ultrastructural layout of specific proteins in health and disease.