Comparison of Staining Methods for Resin-Embedded Peripheral Nerve

Abstract

For routine evaluation of experimental or clinical changes, osmium-impregnated peripheral nerves generally are embedded in epoxy resins and stained with toluidine blue or paraphenylenediamine (PPD). Silver methods, which reveal nerve axons, also have been applied to paraffin or frozen sections for this assessment. In this study, we applied silver staining methods to resin-embedded periphera1 nerves and compared the results with the aforementioned two stains in three laboratory animals. For this investigation, sciatic nerves from rabbits, rats, and pigs were fixed in Zamboni's fluid, embedded in Quetol 65 1 resin, and sectioned at 1–2 μm in an ultramicrotome or a rotary microtome. Plastic sections were treated with sodium ethoxide for removal of resin before staining. The following staining methods were used to evaluate nerve fibers: 1) epoxy stain obtained commercially; 2) 1% aq. PPD solution; 3) silver stain at room temperature with 5% silver nitrate in dark for 30 min followed by reduction in formalin-odium carbonate solution; 4) silver preincubation followed by oven incubation in a developer consisting of hydroquinone, gum mastic and silver nitrate; 5) Bodian protargo1 method. The first silver method (3) showed profiles of myeinated fibers with yellow sharp staining of the myelin sheath. The second silver method with physical developer prouced easily recognizable, discrete brown-to-black profiles of axoplasm of nerve fibers. The Bodian method did not produce consistent results. Silver methods produced better contrast and clear outline of the nerve fibers, whereas toluidine blue or PPD stains produced intense staining but obscuring smaller fibers. This was noticeable in the rabbit and the rat; the pig showed satisfactory results with all four (1–4) methods. The applied silver methods, which seem to be specific for nerve axons, are easy to use and economical and can be used for high-resolution light microscopy and quantitative routine analysis of myelinated fibers from peripheral nerves obtained from nerve biopsies or experimental animals. (The J Histotechnol 27:000, 2004)Submitted December 23, 2003; Accepted May 10, 2004