Em 410

All animal procedures were approved by the NIDDK, National Institutes of Health, Animal Care and Use Committee and were performed in accordance with National Institutes of Health guidelines. The C57BL/6J (Jackson Laboratory) strain was used as an embryo donor. Fertilized oocytes were injected with 100 ng/μl Cas9 mRNA and 50 ng/μl each sgRNA (Synthego) (Fig. 3A). The injected oocytes were transferred to the oviducts of pseudopregnant female mice. Tails from 21-day-old progeny mice were genotyped using forward primer (5′-GTTTTTCTAGGTAAAATTTGCTTTAAAATCGTT) and reverse primer (5′-GGAAAGTTAGTAACAGTTACACATTTAGCTTTG) with PCR conditions as follows: 94 °C for 10 min, 94 °C for 30 s, 64 °C for 15 s, 68 °C for 30 s for 40 cycles. WT allele yielded a 722 bp band, and the KO allele yielded a 655 bp band.
Sciatic nerves harvested from 3-week-old WT and Ahr KO mice were fixed in 2% paraformaldehyde, 2.5% glutaraldehyde, and 0.1 m cacodylate buffer. Sciatic nerves were fixed with 1% glutaraldehyde, 4% paraformaldehyde at 4 °C for 24 h. Ultrathin cross-sections were prepared and analyzed with an electron microscope (Philips EM410). g-ratios were calculated by measuring the mean g-ratio of all of the myelinated axons in three independent microscopic fields with 1.25 × 10³ magnification.

Cells were fixed in 2.5% glutaraldehyde in 0.1M cacodylate buffer, postfixed in 2% osmium tetroxide, dehydrated and embedded in Epon, and examined with a high-voltage electron microscope (Philips EM 410).

Transmission electron microscopical analysis was performed with an EM 410 (Phillips). Samples of 0.5 × 0.5 cm were fixed in buffered glutaraldehyde (2.5%) overnight, post-fixed with OsO4, and then embedded in Agar 100 (Plano), which was let polymerize for at least 24 h. Ultrathin sections were cut with the Ultracut E microtome (Leica). Visualization took place at 80 KV, and images were photodocumented.