Rabbit anti edn3

RNA from head skins was extracted using 0.5 ml of Trizol reagent (Invitrogen) according to the manufacturer’s protocol. RT-PCR was performed according to standard protocols using a reverse transcriptase kit and random primers (Promega). PCR products were size-fractionated by 2% agarose gel electrophoresis. Realtime PCR was performed in triplicate with Power SYBR Green PCR Master Mix on a 7500 Real-Time PCR Detection System (Applied Biosystems). Relative mRNA expression levels were normalized to those of GAPDH and analyzed using the 2-DDCt method. All gene-specific primers were designed by Primer 5 Software. Primer sequences are depicted in Supplementary Table 1.
Dissected head skins were placed in RAPI protein lysis solution (Byotime, China) and lysed on ice for 10 minutes with a Micro Tissue Grinder. Samples were separated by 10% SDS-PAGE and western blots were prepared by standard procedures using either goat anti-TYR (1: 500, Santa Cruz Biotech), mouse anti-β-galactosidase (1: 800, Promega), mouse anti-TYRP1 (1: 500, Abcam) or rabbit anti-EDN3 (1: 1000, Abcam) as primary antibodies and appropriate secondary IRDye® 700CW or IRDye® 800CW-conjugated antibodies. Protein bands were scanned using a LI-COR machine. Mouse anti-α-Tubulin or anti-β-Actin antibodies (1: 2000, Santa Cruz Biotech) were used as internal controls. Protein bands were quantified using Image J software.

The process of X-gal staining has been described^{35 (link)}. For immunofluorescence, 14-µm cryosections were prepared and stained according to standard procedures using the following antibodies as indicated: mouse anti-HMB45 (1: 200, Abcam), mouse anti-TYRP1 (1: 100, Abcam), goat anti-TYR (1: 50, Santa Cruz Biotech), mouse anti-β-galactosidase (1: 100, Promega), rat anti-KIT (1:50, B&D), rabbit anti-Ki67(1: 100, B&D), mouse anti-Ki67 (1: 100, B&D) and rabbit anti-EDN3 (1: 100, Abcam). Appropriate Alexa 488- or 594-conjugated secondary antibodies (Invitrogen) were used at RT for 1 hour. The sections were examined and photographed with a Zeiss fluorescence microscope. For analysis of melanocyte numbers in hair bulbs, 40 µm cryosections were prepared and incubated with rabbit anti-MITF (1: 200, gift from Dr. Arnheiter) primary antibody at 4 °C for 24 hours. Alexa 594-conjugated secondary antibodies (Invitrogen) were used at RT for 2 hours. The sections were examined and photographed with a two-photon Zeiss fluorescence microscope. For quantification of Ednrb-lacZ expression in hair bulb melanocytes, we used Image J software to measure the fluorescence signal. Significance was analyzed using a paired Student’s-t test.