New technique for differential staining of myelinated fibers and nerve cells on paraffin sections.

A simple, rapid method for the differential staining of myelinated nerve fibers and nerve cell bodies, applicable to sections of central nervous system pieces embedded in paraffin, is described. Experimental material fixed by perfusion with mixed aldehydes or necropsy material fixed in formaldehyde can be used. Constant and homogeneous results are obtained with this technique, and the most important characteristic is the absence of differentiation in either of the steps: staining of myelinated fibers and staining of nerve cell bodies. Sections 15 microns thick were attached to slides, dewaxed, and hydrated. After hydration, sections are mordanted (30 min) in 2.5% iron alum (SO4)2FeNH4, and rinsed (1 min) in distilled water. Staining is for 180 min in the following solution: 5 ml freshly made 20% alcoholic hematoxylin diluted with 25 ml of distilled water and 25 ml of absolute ethanol to which 10 ml of 1% Li2CO3 is added. The sections are washed in distilled water (5 min) and stained during 5 min in the following solution: 0.2% pyronine, 20% formaldehyde in distilled water. The sections are dehydrated through 96% and absolute ethanol, cleared in eucalyptol, and mounted in Eukitt. Myelinated fibers appear dark blue, whereas nerve cell bodies are stained red and the cell nucleoli dark blue. This procedure provides an adequate contrast for observation and photography.