, 2001) were maintained on a C57Bl/6 background. Plxnd1+/− mice ( Gu et al., 2005), Npn1 mice ( Gu et al., 2003), and VegflacZ mice ( Miquerol et al., 1999) were maintained on an outbred Swiss Webster background. Sema3e+/− mice were maintained on an 129SVE background. Ngn1 ( Ma et al., 1998) and Ngf ( Crowley et al., 1994) knockout embryos were obtained from Dr. Quifu Ma (Harvard Medical School) and Dr. Rejji Kuruvilla (Johns Hopkins University), respectively. Swiss Selleckchem INCB024360 Webster, C57Bl/6, and 129SVE wild-type mice were obtained from Taconic Farms. All animals were treated

according to institutional and National Institutes of Health guidelines approved by the Institutional Animal Care and Use Committee at Harvard Medical School. Embryos were removed,

immediately frozen in liquid nitrogen, and sectioned at 16 μm using a cryostat (Leica). Sections were fixed in 4% paraformaldehyde (PFA) for 5 min and briefly washed in 1× PBS several times at room temperature. After washing, sections were blocked in 1× PBS containing 0.1% Triton X-100 (PBST) and 10% normal goat serum for 1 hr and then Sirolimus nmr incubated with primary antibodies in blocking buffer at 4°C overnight. For Plexin-D1 immunohistochemisty, embryos were fixed in 4% PFA in 0.1 M phosphate buffer (pH 7.5) for 2 hr and equilibrated in 30% sucrose at 4°C overnight. The antibodies used are: α-neurofilament (1:100; Item No. 2H3, Developmental Studies Hybridoma Bank), α-PECAM (1:500; 553370, BD Pharmingen), α-Plexin-D1 (1:6,000, a gift from Dr. Yutaka Yoshida, Cincinnati Children’s Hospital), and α-TrkA (1:500; 06-574, Millipore). After washing in 1× PBST for 5 min three times, sections were incubated in Alexa Fluor-conjugated secondary antibodies (1:1,000, Invitrogen) for 1 hr, then washed several times in PBST, and mounted with Fluoromount G (Electron Microscopy Sciences). Immunostained sections were analyzed by fluorescence microscopy using a Nikon Eclipse 80i microscope equipped with a all Nikon DS-2 digital camera. Images were processed using Adobe Photoshop and ImageJ (National Institutes

of Health). For whole-mount whisker follicle staining, embryos were fixed in 4% PFA for 6 hr and equilibrated in 30% sucrose at 4°C overnight. Embryo snout areas were sectioned at 100 μm using a cryostat (Leica) and blocked in 1× PBST, 10% DMSO, and 5% normal goat serum for 1 hr, and then incubated with primary antibodies in blocking buffer at room temperature for 2 days. The antibodies used were α-neurofilament (1:50; 2H3) and α-VE-cadherin (1:100; ab33168, Abcam). After washing in 1× PBST for 1 hr five times, sections were incubated in Alexa Fluor-conjugated secondary antibodies (1:500, Invitrogen), diluted in the blocking buffer for 1 day, then washed several times in PBST, and dehydrated with 100% methanol. Before mounting, sections were cleared with benzyl alcohol and benzyl benzoate mixture.