A Batch Staining Method for Brain Slices Allowing Volume Measurements of Grey and White Matter Using an Image Analyzing Computer (Quanttmet 720)

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Abstract
Normal formalin-fixed gelatin-embedded cerebral hemispheres were serially sliced and the 25 to 30 slices from each hemisphere were batch stained by a modification of Mulligan's method. Following washing in water the slices were immersed in Mulligan's acid/copper sulfate/ phenol solution for 20 minutes at room temperature, treated with a xylene/Polyclens mixture for 20 seconds and immediately transferred to a 2% sodium hydroxide solution for 10 seconds. Final staining was by immersion in 2% potassium ferrocyanide which was followed by washing in tap water. The grey matter was stained a brick red color while the whiteness of the white matter was accentuated. Following staining the slices were stored between sheets of black paper in 2% aqueous formalin prior to measurement of the respective areas of grey and white matter using a Quantimet 720 image analyzing computer. The method is rapid and color stable, and reduces the risk of exposure to toxic fumes by eliminating the need for hot phenol solutions. This technique is also suitable for the macroscopic demonstration and quantitation of demyelinating conditions in the brain.