Chicken β tubulin 3

Mice were anesthetized and perfused with 4% formaldehyde in 0.1m phosphate‐buffered saline (PBS, pH 7.4). DRGs were dissected rapidly, postfixed in the same fixative solution at 4 °C for 4 h, and cryoprotected in 30% sucrose overnight. A series of 20‐µm transverse sections were cut on a cryostat. The sections were blocked with 5% goat serum and 0.3% TritonX‐100 in 0.01 m PBS at room temperature for 1 h and then incubated with rabbit anti‐Pou4f3 (1:800, GeneTex) plus biotinylated IB4 (1:200, Sigma), mouse anti‐CGRP (1:200, Abcam), mouse anti‐NF200 (1:200, Sigma), mouse anti‐glutamine synthetase (1:1,000, Sigma) or chicken β‐tubulin III (1:500, EMD Millipore) or with rabbit anti‐RALY (1:2,500, Abcam) plus mouse anti‐glutamine synthetase (1:1,000, Sigma) or chicken β‐tubulin III (1:500, EMD Millipore) at 4 °C over‐two nights. The sections were then incubated with species‐appropriate Cy2‐ or Cy3‐conjugated secondary antibody (1:500, Jackson ImmunoResearch), or FITC‐labeled Avidin D (1:200, Sigma) at room temperature for 2 h. Control experiments were performed in parallel. The images were captured with a Leica DMI4000 fluorescence microscope (Leica).