Table 2.

Sihler staining protocol for nerve mapping

Processing step	Duration	Target reaction
4% paraformaldehyde; change solution once a week	50–55 days	Tissue fixation
3% potassium hydroxide (KOH) with 3 drops of 3% v/v hydrogen peroxide per 100 mL; change solution twice a week	50–60 days	Maceration and depigmentation. Endpoint specimen becomes translucent and nerves can be clearly seen as white fibers under a transilluminated microscope
Sihler I solution; change solution twice a week	30 days	Decalcification
Sihler II solution; change solution every other week or more often if solution changes from dark blue to purple	30–40 days	Staining. Endpoint nerves become dark blue
Sihler I solution (with agitation); change solution 1–2 times as needed when it becomes blue or purple	24 h	Destaining. Endpoint nerves become dark purple but all other tissues become a transparent lavender color under a transilluminated microscope; check every 2–3 h
0.05% lithium carbonate (with agitation)	2 h	Neutralizing. Endpoint Nerves turn from purple to deep blue; check every 30 min under a transilluminated microscope
50% glycerine	4 days	Clearing. Endpoint when the finest nerve twigs can be seen clearly under a transilluminated microscope; check daily
100% glycerine with thymol crystals	n/a	Long-term preservation

Perform water washes between steps: rinse 5 times, incubate with agitation for 1 h, then rinse again 5 times