Vectashield hard set mounting medium h 1400

A custom DNSP-11 polyclonal antibody (see below) was used for immunohistochemistry, immunoprecipitation, and ELISA. Tyrosine hydroxylase antibody was purchased from Millipore Chemicon (MAB318, Millipore Chemicon, Temecula, CA). GDNF antibody was purchased from R&D Systems (AF-212-NA, R&D Systems, Minneapolis, MN). Secondary antibodies include peroxidase-conjugated anti-rabbit IgG (PI-1000, Vector Laboratories, Burlingame, CA), Alexafluor anti-goat 568 and Alexafluor anti-rabbit 488 fluorescent (Life Sci. Invitrogen Molecular Probes). VectaShield H-1400 Hard-Set Mounting medium for coverslipping dual-fluorescent specimens was used (Vector Laboratories, Burlingame, CA). Fluorescent images acquired on a Nikon microscope. Refrigerators and freezers were continuously monitored for temperature variations. Other reagents were purchased from Fisher Scientific unless otherwise specified. DNSP-11 peptide synthesis and purification were performed by GenScript (Piscataway, NJ USA). Peptides were verified as >98% pure upon receipt via reverse-phase HPLC, LC-MS, and amino acid sequencing, as previously reported (Bradley et al., 2010 (link)).

We processed a 1-in-4 series of AI, vmPFC, PVT, BLA, and vSub for immunohistochemical detection of Fos-protein and CTb. We rinsed free-floating sections (3×10 min) and then incubated them in 10% NHS with 0.5 % PBS-Tx for 2 h. We then incubated the sections for at least 48 h at 4 °C in 0.5 % PBS-Tx containing 2% NHS and rabbit anti-c-Fos primary antibody (1:8000, Cell Signaling Technology, Phospho-c-Fos, 5348S; RRID: AB_10013220) and goat anti-CTb primary antibody (1:5000, CTb 703; List Biological Laboratories; RRID: AB_10013220). We rinsed the sections in PBS and incubated them for 3 h in PBS containing 2% NHS and donkey anti-rabbit Alexa Fluor 594 (711-585-152; RRID: AB_2340621; Jackson ImmunoResearch) and donkey anti-goat Alexa Fluor 488 (705-546-147; RRID: AB_2340430; Jackson ImmunoResearch) diluted to 1:2000. We rinsed the sections in PBS (3 × 10 min) and mounted them onto gelatin-coated slides, partially dried, and coverslipped the sections with Vectashield Hard Set Mounting Medium (H-1400; Vector Laboratories).