Fixation and staining methods for macroscopical investigation of the brain

Abstract

The proper preservation of human brain tissue is an indispensable requirement for post-mortem investigations. Neuroanatomical teaching, neuropathological examination, neurosurgical training, basic and clinical neuroscientific research are some of the possible downstream applications of brain specimens and, although much apart from one another, proper tissue fixation and preservation is a common denominator to all of them. In this review, the most relevant procedures to fixate brain tissue are described. In situ and immersion fixation approaches have been so far the most widespread ways to deliver the fixatives inside the skull. Although most of them rely on the use of formalin, alternative fixative solutions containing lower amounts of this compound mixed with other preservative agents, have been attempted. The combination of fixation and freezing paved the way for fiber dissection, particularly relevant for the neurosurgical practice and clinical neuroscience. Moreover, special techniques have been developed in neuropathology to tackle extraordinary problems, such as the examination of highly infective specimens, as in the case of the Creutzfeldt-Jakob encephalopathy, or fetal brains. Fixation is a fundamental prerequisite for further staining of brain specimens. Although several staining techniques have been developed for the microscopical investigation of the central nervous system, numerous approaches are also available for staining macroscopic brain specimens. They are mostly relevant for neuroanatomical and neuropathological teaching and can be divided in white and gray matter staining techniques. Altogether, brain fixation and staining techniques are rooted in the origins of neuroscience and continue to arouse interest in both preclinical and clinical neuroscientists also nowadays.

Beyond the surface: how ex-vivo diffusion-weighted imaging reveals large animal brain microstructure and connectivity

“…All these factors are usually understood as a whole under the umbrella term of PMI, although wide variations can occur (e.g., a 24 h at 35°C ambient temperature is different in outcome from 24 h at 5°C), although a recent study seems to indicate minimal immunohistochemical effects ( Koehler et al, 2024 ), and in details, PMI explains only part of the problems encountered in large brain fixation. For all these reasons, fixation by immersion seems to remain the most used alternative, as is the case in human brain preservation ( Nardi et al, 2023 ). The fixative should be changed after the first 24 h, and a large volume is necessary (ideally 10 times the volume to be fixed).…”