Rabbit anti c kit

Adult mice were euthanized with CO2. Hairy skin from the top of each foot was removed and fixed in 100% acetone for 8 hours at room temperature, followed by immersion in 1mg/ml X-gal in staining solution (0.1 M Phosphate Buffer pH 7.5, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 20 mM Tris-HCL, 0.02% NP-40, 0.01% sodium deoxycholate) for 48 hours at 4°C. Tissues were then rinsed, post-fixed in 4% paraformaldehyde/1xPBS overnight, washed in PBS, submerged in 30% sucrose/1xPBS, and embedded in O.C.T. media. Sections of 15–20 μm thickness were cut and collected on glass slides and kept frozen until use. Antibodies on these sections included Rat anti-CD3 epsilon (1:100, NBP1-26685, Novus Bio) and Rabbit anti-C-Kit (1:100, D13A2, Cell Signaling Tech). For DRG neurons, whole ganglia were briefly fixed in 4% paraformaldehyde for 5 minutes, followed by X-gal staining as above for 1.5–2 hours, and embedded for cryosections as above. Sections (10 μm thickness) were counterstained with Rabbit anti-Tuj1 (1:1000, Sigma T2200) and DAPI. Brightfield and fluorescence images were acquired on a Zeiss Observer V1. Tuj1-positive neurons with nuclei in the plane of sectioning were counted manually, and percent overlay with X-gal staining were calculated.