Rabbit anti six1

E9.5 and E10.5 embryos were immersion fixed and prepared for cryosectioning and antibody labeling as described previously (Karpinski et al., 2016 (link)). At P8, each CNgV was dissected after aldehyde perfusion fixation, and then prepared and embedded whole for cryosectioning. The primary antibodies used were mouse anti-βIII tubulin (BioLegend, 801201, 1:1000), rabbit anti-Six1 (Proteintech, 10709, 1:1500), rabbit anti-fibronectin (Millipore, AB2033, 1:1000), anti-cleaved caspase 3 (Cell Signaling Technology, 9661, 1:200), chicken anti-GFP (Abcam, ab13970, 1:1000), mouse anti-NeuN (Merck Millipore, MAB377, 1:1000), rabbit anti-NeuN (Cell Signaling Technology, 24307, 1:400), mouse anti-BrdU (BD Biosciences, 555627, 1:100), rat anti-BrdU (Novus, NB500-169, 1:100), rabbit anti-Sox2 (Stemgent, 09-0024, 1:100), goat anti-TrkB (R&D Systems, AF1494, 1:100), anti-TrkA (Alomone Labs, ANT-018, 1:100), goat anti-Ret (Neuromics, GT15002, 1:50) and rabbit anti-TrpV1 (Alomone Labs, ACC-030, 1:100). Primary antibody labeling was visualized using Alexa Fluor 488-, 54- or 647-conjugated secondary antibodies (Molecular Probes, 1:4000, 488; 1:2000, 546 and 1:1000, 647). Standard BrdU immunolabeling techniques were used after acid treatment for antigen retrieval. Images were collected on a Leica Tiling or a Zeiss 710 confocal microscope.

The whole cell protein was extracted using RIPA buffer combined with protease inhibitor (Merck Millipore). The protein concentration was determined by the Bradford method (Beyotime, Shanghai, China), and 20 μg of protein was separated by 10% SDS/PAGE (Beyotime) and transferred to polyvinylidene fluoride membrane (Merck Millipore). Then, the primary antibodies were incubated at 4 °C overnight and secondary antibodies for 1 h at room temperature. The primary antibodies used were mouse anti‐β‐actin (Sigma, USA), rabbit anti‐SIX1 (Proteintech, Rosemont, IL, USA), PCNA, c‐myc, cyclin‐D1 and cyclin‐A1 (Cell Signaling Technology, USA).