Alexa fluorescent conjugated goat or donkey secondary antibodies

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).

Spinal columns of embryos and neonatal mice at the desired developmental stages were dissected out and directly immersed in PBS/4% paraformaldehyde (PFA) for 2 to 4 hr at 4°C. Postnatal mice were sacrificed with CO₂, transcardially perfused with 4% PFA, and spinal columns were dissected out and post-fixed with 4% PFA for 2 hr at 4°C. They were then cryo-protected in 1× PBS, 30% sucrose overnight. 20-µm frozen sections of SC and DRGs were cut using a CM1950 cryostat (Leica, Buffalo Grove, IL). Immunostaining of SCs and DRG sections were performed as described previously (Niu et al., 2013 (link)). Antibodies used are as follows: rabbit anti-GFP (A-11122, 1:2000, Invitrogen, Carlsbad, CA), chicken anti-GFP (GFP-1020, 1:1000, Aves, Tigard, OR), chicken anti-NF200 (NF-H, 1:500, Aves), rabbit anti-NF200 (N4142, 1:1000, Sigma, St. Louis, MO), rabbit anti-cRet (18121, 1:50, IBL, Minneapolis, MN), rabbit anti-NTRK1 (06-574, 1:1000, Millipore, Temecula, CA), guinea pig anti-VGLUT1 (AB5905, 1:1000, Millipore), rabbit anti-phospho-S6 (2215s, 1:200, Cell Signaling, Beverly, MA), and Alexa Fluorescent conjugated Goat or Donkey secondary antibodies (1:500, Invitrogen or Jackson ImmunoResearch, West Grove, PA).