The Use Of Nigrosin Staining To Increase Brain Slice Contrast For Neuroanatomy Teaching

Although plastic models and electronic resources play an important role in demonstrating concepts in neuroanatomy, the study of human cadaver brain remains important where it is necessary to illustrate detail, spatial relationships, or in clinical teaching. The most difficult structures to visualize in embalmed brains are the deep cerebral and cerebellar nuclei due to the muted contrast after embalming. Many contrast enhancing staining protocols have several drawbacks including vague published protocols, variability of staining, the need for counter‐stains, or the length of the staining process. The purpose of our study was to evaluate several simple stains and protocols that would be effective in increasing grey matter contrast in brain slices. Whole brains were extracted from cadavers fixed with 65% ethanol, 10% propylene glycol, 4% phenol, 4% Dettol and 2% formalin after 10–12 months. Brains were postfixed with 10% formalin for 2–3 weeks before use. Two Plexiglas frames were constructed with horizontal (transverse) or vertical (coronal) slits spaced 1 cm apart to facilitate the cutting of serial sections. Serial transverse or coronal slices of brains were washed in tap water for 1 min prior to staining. Brain slices were stained in various dilutions of aqueous toluidine blue, cresyl violet, or Nigrosin. The contrast between grey and white matter was measured with a standardized camera and image analysis was performed on 8‐bit images using Image‐J. Overstained or understained sections were obtained with all protocols except Nigrosin. The most successful staining was obtained on sections stained with 0.5% aqueous Nigrosin for 15 min followed by clearing in a series of water and ethanol rinses. Sections were stored on a stainless steel tray covered with a moist cloth for various times before quantitative analyses was performed. The depth of staining was evaluated in sections cut tangential to the stained surface. The robustness of the staining was evaluated in sections stored in water, 2% aqueous glycerol, 100% ethanol, or 100% acetone for various times up to 4 wks. We conclude that the staining of brain sections with Nigrosin provides high contrast differentiation of grey and white matter. Interestingly, details of white matter tracts became visible but certain deep brain nuclei were consistently unstained. There was minimal fading of the staining after storage in organic and inorganic solvents indicating that the method may be useful for long term storage as both wet or plastinated specimens. Nigrosin staining of brain slices serves as a simple method to increase the visibility of brain structures for the study of neuroanatomy. Support or Funding Information Education Program in Anatomy, McMaster University