Immunohistochemistry of Cutaneous Nerves

Mice were euthanized with CO₂, transcardially perfused with PBS/4% paraformaldehyde (PFA). Back hairy skin tissue (de haired with Nair) and footpad of hind paw were dissected and post-fixed with 4% PFA for 2 hours at 4°C, and cryo-protected in 30% sucrose in 1XPBS overnight. Frozen sections of 40μm were cut using a Leica CM1950 cryostat. Immunostaining of sections, and whole mount hairy skin were performed as described previously [24] (link), [25] (link). Antibodies used are as follows: rat anti-cytokeratin 8 (DSHB, TROMA1, 1∶20), goat anti-TrkC (R&D, AF1401, 1∶1000), chicken anti-GFP (Aves, GFP-1020, 1∶1000), rabbit anti-GFP (Invitrogen, A-11122, 1∶2000), chicken anti-NFH (Aves, NF-H, 1∶1000), rabbit anti-NFH (Sigma, N4142, 1∶1000), and Alexa Fluorescent conjugated Goat or Donkey secondary antibodies (Invitrogen or Jackson IR, 1∶500-1000).