Ab144p

Primary antibodies used in this work were: rabbit anti-RFP (1:2000, ABIN129578), goat anti-RFP (1:100, AB0040-200), chicken anti-GFP (1:2000, Aves GFP 1020), goat anti-Iba1 (1:200, NB 100-1028), rabbit anti-GFAP (1:500, Dako-Z0334), rat anti-FLAG (1:500, 637304), rabbit anti-RIG-I (1:100, ab45428), rabbit anti-NF-kB (1:400, 8242), rabbit phospho p38 MAPK (1:500, 4511L) and Chat (1:1000, Chemicon AB144P). The secondary antibodies used in this work were: donkey anti-chicken AF488 (1:1000, 703-545-155), donkey anti-rat AF488 (1:1000, A21208), donkey anti-rabbit Cy3 (1:1000, 711-165-152), donkey anti-goat Cy3 (1:1000, 705-165-147), donkey anti-goat Cy5 (1:1000, 705-175-147), donkey anti-rabbit Cy5 (1:1000, 711-175-152), goat anti-rat Cy5 (1:1000, 712-005-153).
All incubation steps were conducted at 4°C on a rocking platform for 24-48h and all antibodies were diluted in blocking solution (1% bovine serum albumin and 0.3% Triton X-100 in PBS) except for rabbit RIG-I which was diluted in 3% BSA, 10% donkey serum, 0.3% Tx. Between primary and secondary antibody staining, samples were washed four times with PBS at room temperature.

Sections processed for ChAT immunocytochemistry were rinsed with 3× 10 min PB, followed by 15 minutes incubation in 0.3% H₂O₂, followed by 1 hour incubation in PB supplemented with 4% bovine serum albumin (BSA) and 0.3% Triton X-100. The sections were then incubated overnight at 4°C with anti-ChAT goat polyclonal antibody (Chemicon, Temecula, CA, AB144P; dilution 1:100). After rinsing with 3×10 min PB, the sections were incubated for 1 hour at room temperature in a 1:200 dilution of biotinylated secondary antibody (Vector Laboratories, Burlingame, CA), rinsed with PB, and incubated with avidin-biotinylated horseradish peroxidase for 1 hour. The sections were then rinsed and the peroxidase reaction was developed with 0.05% 3,3-diaminobenzidine-4 HCl (DAB) and 0.03% hydrogen peroxide (H₂O₂). Finally, the sections were mounted on gelatin-coated slides and air-dried for inspection. Processed sections were viewed, analyzed, and photographed with bright field microscopy using Surveyor with Turboscan software (Objective Imaging, Cambridge, UK) at 20×.

Anti-Chat goat polyclonal antibody (Chemicon, AB144P, RRID:AB 11214092) was raised against human placental choline acetyltransferase. On Western blot with mouse brain lysate the antibody stains a single band of 68–70 kDa, apparent molecular weight for Chat (manufacturer’s technical data). Antibody specificity on tissue sections was shown by matching localizations of Chat:GFP signal with immunohistochemistry signal by Mesnage et al., 2011 (link).

To determine the transmitter phenotype of dPSNs, we performed experiments with FG retrograde labeling combined with immunohistochemistry. Four days after transection injury, animals received a lethal dose of ketamine/xylazine and were perfused transcardially with a fixation solution containing 4% paraformaldehyde and 0.3% glutaraldehyde in 0.1 M phosphate buffer at pH 7.4. T7–9 spinal cords were removed from the spines and cryocut into 20-μm thick transverse sections. We collected sections at every 100 μm interval as a set for each neurotransmitter staining. Immunohistochemistry was performed using antibodies raised against protein-conjugated amino acid neurotransmitters (anti-glutamate, G9282, 1:10,000, Sigma-Aldrich; anti-GABA, A2052, 1:5,000, Sigma-Aldrich; anti-glycine, 1:2,000, AB139, Chemicon, Temecula, CA; Millipore anti-Choline acetyltransferase, 1:100, AB144P, Chemicon) producing staining of distinct cellular populations. After incubating spinal cord sections with the primary antibody or antibodies for 48 hours, secondary antibodies matching the species of the primary antibody and labeled either with Cy3 (ab6939 Abcam MA) or Alexa Fluor 488 (A-11001 Life Technologies, NY) were employed to visualize their binding sites. To test the specificity of the primary antibody, they were omitted from control sections and no immunoreactivity was detected.