IHC staining of rat spinal cord dorsal horn: Antiserum was used on perfusion fixed tissue. Perfusion: 1) calcium-free Tyrode’s solution, 2) paraformaldehyde-picric acid fixative, and 3) 10% sucrose in PBS as a cryo-protectant. Desired tissues were dissected and stored overnight in 10% sucrose in PBS. Slide-mounted tissue sections were processed for indirect immunofluorescence. Slides were incubated with blocking buffer for 1 hour at room temperature. Primary antiserum was diluted with blocking buffer to the appropriate working concentration. Blocking buffer was removed and slides were incubated for 18-24 hours at 4oC with primary antiserum. Slides were rinsed 3 times and then incubated with secondary antibodies for 1 hour at room temperature. Slides were again rinsed 3 times and coverslipped. Staining was examined using fluorescence microscopy.