Mouse anti mitf

Immunohistochemistry of coronal cryosections was performed as previously described (Burnett et al., 2017 (link)). Primary antibodies were used at the following concentrations: rabbit anti-PAX2 (1:450, Covance Research Products), mouse anti-PAX6 (1:50, Developmental Studies Hybridoma Bank), sheep anti-CHX10 (1:600, Exalpha Biologicals), mouse anti-MITF (1:1500, Abcam), rabbit anti-OTX2 (1:600, Upstate Biotechnology), mouse anti-COUPTFII (1:300, R & D Systems), goat anti-SOX1 (1:100, R & D Systems), rabbit anti-SOX2 (1:300, EMD Millipore), mouse anti-γ-TUBULIN (1:1000, Sigma-Aldrich), rabbit anti-ARL13B (1:3000, T. Caspary, Emory University). Images were taken with either a Zeiss Axioplan 2 or a Keyence BZ-×710 fluorescence microscope. High-magnification images of cilia are maximum intensity projections from z-stacks taken on a Zeiss LSM510 confocal microscope.

Immunofluorescence was performed as published (Rabbani et al., 2011 (link)). Briefly, skin tissues were embedded in paraffin and cut into 6um sections. Tissue sections were incubated with primary antibodies listed below for 2 hours at room temperature or overnight at 4°C, followed by incubation with Alexa 488/594-conjugated secondary antibodies (1:200; Invitrogen, Carlsbad) for 1 hr at room temperature. Sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame). The primary antibodies used were: mouse anti-β-catenin (1:400, Sigma-Aldrich, c7207), mouse anti-MITF (1:100, Vector Laboratories, VP-M650) (used in Figure 3–4), mouse anti-MITF (1:100, Abcam, ab12039) (used in Figure 1), goat anti-sox10 (1:100, Santa Cruz Biotechnology, sc-17342), goat anti-Dct (1:100, Santa Cruz Biotechnology, sc-10451), rabbit anti-S100 (1:100, DAKO, Z0311) and rabbit anti-Tcf1 (1:100, Cell Signaling Technology, C63D9). Images were taken with an inversed Eclipse Ti microscope (Nikon, Japan) or an upright Axioplan microscope (Zeiss, Germany).