Encephalic Gray Matter macroscopic staining. An historical overview and three techniques comparison

Abstract

Macroscopic staining in central nervous system anatomical specimens is a technique that allows obtaining better differentiation of multiple structures as the basal ganglia, thalamus, putamen, globus pallidus amongst others. The methods for staining are based on using the different components of the brain, mainly the lipids in white matter. When staining is pursued, these lipids join with reactants such as phenol, generating a “coat” that prevents the staining of white matter during the procedure, restricting it to the grey matter. The original process described by Mulligan can be modified with multiple reactants to generate a broad range of colors. Our objective is to describe the technical differences between three broadly used staining protocols: Roberts, Mulligan and Prussian Blue comparing the results between them according to the quality of the reactants used. A literature review in Medline for articles published in English until October 2017 was done. Regular terms were used, including: Brain, Encephalic, Cerebrum, Staining, Stained. And Medical Subject Headings, including: Brain [title] OR encephalic [title] OR cerebrum [title]) AND (staining [title] OR stained [title] OR stain [title] OR Staining and Labeling [majn]). A total of 511 articles were obtained. 484 were excluded and a total of 28 articles were included. For staining trials, three human brains from our laboratory collection previously preserved in 10% v/v formaldehyde aqueous solution were selected. Coronal and axial cuts were performed, and staining was randomly performed in the slices following the instructions of the original protocols proposed for each staining in two randomly created specimen’s groups: USP quality reactants and Analytical grade reactants. An additional modification was the use of tap water vs distilled water. The most important independent variable for us was a room temperature of 12 C. All the methods were replicable with good results. Technically, Prussian Blue presented a greater risk of white matter staining. However, this problem could be easily solved by extended immersion of 3 to 4 minutes in Mulligan’s solution rather than the original protocol for Mulligan’s Staining that suggest 2 minutes. In addition, the use of tap water instead of distilled water did not change results. Finally, according to our experience, the purity of the reactants can be lower than suggested in old protocols; comparisons between results of high‐quality reactants vs USP quality did not exposed significantly interference with the outcomes.