Fig. 2. Perineurial cells are not neural crest derived. Sciatic nerves from Wnt1-Cre⁺loxpRosa⁺ mouse pups were dissected and processed for X-gal staining (A), immunohistochemistry (B), or electron microscopy (C,D), at either postnatal day 3 (A) or 11 (B-D). According to each technique the perineurial cells failed to express ß-gal and therefore were not neural crest derived. Sections through X-gal stained (A) or anti-ß-gal antibody stained (B) nerves consistently exhibited strong staining throughout the endoneurial space, but the flattened layers of perineurial cells that distinguish different bundles of axons were notable for their lack of X-gal or anti-ß-gal antibody staining (arrows, A,B). We also failed to observe X-gal staining in perineurial cells from P₀-Cre⁺ß-actin-lacZ⁺ mice or Wnt1-Cre⁺ß-actin-lacZ⁺ mice (data not shown). Note that the red fluorescent cell in the perineurial layer (bottom right corner; B) was an autofluorescent blood cell within a blood vessel (see Fig. S4 in supplementary material). Bluo-gal forms an electron dense precipitate that is visible as small black crystals (arrowheads, C) by electron microscopy after digestion by ß-galactosidase (Weis et al., 1991). Bluo-gal staining was consistently absent from perineurial cells (P; panel C), but was visible in most Schwann cells (to the right of the perineurial cells in panel C). The myelin sheaths within the Schwann cells appear light and fragmented because bluo-gal staining required fixation conditions that did not effectively preserve myelin. Note the lack of bluo-gal staining in a nerve section from a control littermate (D).