Aurora Prado-Sanhueza; Carlos Veuthey; Daniel Vargas; Ignacio Roa; Pablo A. Lizana; Telma Masuko & Nicolás E. Ottone

Effective visualization of central nervous system structures is essential for neuroanatomical education, particularly at the undergraduate level where access to fresh specimens and advanced imaging resources is often limited. Classical staining methods such as those developed by Sincke, Mulligan, and Braak have contributed significantly to anatomical teaching; however, many of these protocols involve complex procedures, hazardous chemicals, or prolonged fixation and processing times that hinder their widespread application in educational contexts. In response to these limitations, this study aimed to develop and evaluate a simplified, low-toxicity adaptation of the Mailland technique for staining formalin-fixed brain tissue. Two bovine (Bos taurus) brains were used, sectioned parasagittally into 18 slices of 3 mm thickness, and stained with ferric chloride and potassium ferrocyanide at varying concentrations (1%, 0.75%, and 0.5%) and immersion times. The resulting blue pigmentation allowed clear differentiation between gray and white matter, with staining intensity increasing proportionally with concentration and exposure time. Group 1 (1% for 5 min and 1 min, respectively) exhibited the most intense coloration, while Group 6 (0.5% for 1 min and 30 s) still demonstrated a reliable contrast, highlighting the method's efficiency even under minimal conditions. The adapted protocol requires no specialized equipment, operates at room temperature, and uses readily available reagents, making it highly accessible for academic laboratories with limited infrastructure. Compared to other classical methods, our approach is faster, more economical, and safer, with consistent results across slices. This technique, therefore, provides a valuable pedagogical tool for enhancing neuroanatomical education through improved structural visualization.

KEY WORDS: Brain staining; Mailland technique; Neuroanatomy; Anatomical education.

PRADO-SANHUEZA, A.; VEUTHEY, C.; VARGAS, D.; ROA, I.; LIZANA, P.A.; MASUKO, T. & OTTONE, N.E. Adaptation of the Mailland technique for neuroanatomical staining: a cost-effective and accessible method for teaching brain structure. Int. J. Morphol., 43(6):1869-1878, 2025.