3A. H&E staining of enlarged right sciatic nerve where Nf1-/-;LacZ+ SKPs had been implanted (b: original magnification 20X; d: original magnification 40X) showing features consistent with plexiform neurofibroma, including disordered, convoluted bundles of cells exhibiting spindle-cell morphology, with ovoid and spindle-shaped nuclei and adjacent fine fibrillar stroma. There is also massive mast cell infiltration (black arrows) within the tumor as demonstrated by Leder staining (f), which stains mast cells red. As shown by H&E staining, these features are absent in the left sciatic nerve where control Nf1+/-;LacZ- SKPs were implanted (a: original magnification 20X; c: original magnification 40X) and Leder staining (e).
3B. SKPs were purified from skin taken from neck, ears, or back of CMV-CreERT2;Nf1flox/-;ROSA26flox-stop-flox mice and exposed to 4OH-tamoxifen to induce recombination at the ROSA26 and Nf1 loci. Two to three months after SKP implantation, mice underwent total body perfusion with 4% paraformaldehyde and left and right sciatic nerves were harvested for gross X-gal staining of whole nerves. The nerves were then post-fixed in formalin for paraffin sectioning and subsequently counter-stained with nuclear fast red. Representative X-gal stainings of whole left and right sciatic nerves show that the enlarged right sciatic nerve is X-gal positive (c, right panel) but not the control left sciatic nerve (c, left panel). The corresponding paraffin sections also show histological evidence of neurofibroma with X-gal-positive, disordered spindle-cell morphology, with ovoid and spindle-shaped nuclei for the right sciatic nerve (b) but not the control left sciatic nerve (a). The left and right sciatic nerves were also subjected to semiquantitative PCR genotyping (d). The recombined loxP sites (Δfloxed Nf1) at the Nf1 locus were detected in the tumor on the right sciatic nerve (d), further indicating that the neurofibromas are derived from the implanted SKPs. (Flox Nf1 = floxed Nf1 allele; Nf1 KO = mutant Nf1 allele).