A simple protocol for paraffin‐embedded myelin sheath staining with osmium tetroxide for light microscope observation

Abstract

Experimental investigation of peripheral nerve fiber regeneration is attracting more and more attention among both basic and clinical researchers. Assessment of myelinated nerve fiber morphology is a pillar of peripheral nerve regeneration research. The gold standard for light microscopic imaging of myelinated nerve fibers is toluidine blue staining of resin-embedded semithin sections. However, many researchers are unaware that the dark staining of myelin sheaths typically produced by this procedure is due to osmium tetroxide postfixation and not due to toluidine blue. In this article, we describe a simple pre-embedding protocol for staining myelin sheaths in paraffin-embedded nerve specimens using osmium tetroxide. The method involves immersing the specimen in 2% osmium tetroxide for 2 h after paraformaldehyde fixation, followed by routine dehydration and paraffin embedding. Sections can then be observed directly under the microscope or counterstained using routine histological methods. Particularly good results were obtained with Masson's trichrome counterstain, which permits the imaging of connective structures in nerves that are not detectable in toluidine blue-stained resin sections. Finally, we describe a simple protocol for osmium etching of sections, which makes further immunohistochemical analysis possible on the same specimens. Taken together, our results suggest that the protocol described in this article is a valid alternative to the conventional resin embedding-based protocol: it is much cheaper, can be adopted by any histological laboratory, and allows immunohistochemical analysis to be conducted.

Use of poly(DL-lactide-ε-caprolactone) membranes and mesenchymal stem cells from the Wharton's jelly of the umbilical cord for promoting nerve regeneration in axonotmesis: In vitro and in vivo analysis

“…Fixation was carried out using 2.5% purified glutar-aldehyde and 0.5% saccarose in 0.1 M Sorensen phosphate buffer for 6-8 hours and resin embedding was obtained following Glauerts' procedure ( Scipio et al, 2008). Series of 2-mm thick semi-thin transverse sections were cut using a Leica Ultracut UCT ultramicrotome (Leica Microsystems, Wetzlar, Germany) and stained by Toluidine blue.…”

The sciatic nerve injury model in pre-clinical research

“…To reach this goal, whereas several histochemical and immunohistochemical methods have proven to be useful (Di Scipio et al, 2008;Sinis et al, 2009;Carriel et al, 2014) , the far most used method is toluidine blue staining on semithin sections from osmium post-fixed and resin-embedded blocks ). This method allows high resolution imaging of myelinated nerve fibers.…”

Repairing nerve gaps by vein conduits filled with lipoaspirate-derived entire adipose tissue hinders nerve regeneration

“…Specimens were immediately "xed in a solution containing 2.5% glutaraldehyde and 0.5% sucrose in 0.1 M Sö rensen phosphate buffer (pH 7.2) for 4…6 h . The specimens were then washed in a solution containing 1.5% sucrose in 0.1 M Sö rensen phosphate buffer for a period of 6…12 h, post-"xed in 2% osmium tetroxide, dehydrated and embedded in Glauerts• embedding mixture (Di Scipio et al, 2008).…”