Whole-mount tail skin staining and imaging

1. Harvest the entire tail skin and flatten the skin in a dish. 
2. Cut the tail skin into 0.5×0.5 cm pieces and flatten them in the dish for 5 min. 
3. Place the skin piece in 4 mL 20 mM EDTA (pH 8.0) solution in 5 mL tube and incubate it at 37℃ with shaking at 80 rpm for 2h. 
4. Carefully remove the epidermis from dermis in the anterior-posterior direction with a fine-tipped tweezer under stereoscope.
5. The epidermis was then flattened and fixed for 10 min in 4% PFA in 24 well dish. 
6. Wash whole-mount skin samples in PBS, 3×45 min at RT.
7. Remove PBS and add blocking buffer for 30 min at RT with shaking.
8. Remove blocking buffer and add primary antibodies in blocking solution for 8 hours at 4⁰C on a shaker.
9. Remove primary antibodies and wash whole-mount skin samples with PBS, 3×15 min at RT.
10. Remove PBS and add secondary antibodies in blocking solution for 8 hours at 4⁰C on a shaker.
11. Remove secondary antibodies and wash whole-mount skin samples with PBS, 3×15 min at RT.
12. Mount sections on to slides using glycerin + DAPI and secure with cover glass.
13. For the whole-mounts of tail skin, images were acquired using a 20×0.75 objective lens. Z-stacks were acquired at a resolution of 1024×1024, or 512×512 for large samples. Tissue samples were imaged on a Nikon A1-R confocal microscope. 
14. Microscopy data were analyzed using Imaris (3D software) with the 3D visualization module. Panels were labeled with Adobe Illustrator CS6.

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