Substance P Antibody

We have successfully used the rabbit anti-substance P-antibody (Immunostar #20064) to examine the distribution of substance P in the brain of adult zebrafish. Zebrafish Brain Preparation: We perfused zebrafish with PBS and 4% paraformaldehyde and removed the brain after postfixation with 4% paraformaldehyde for two days. Brains were embedded and frozen in OCT and subsequently cut using a Leica cryostat. Immunofluorescence staining. DAY 1 1. Slides are dried at room temperature for at least 30 min. 2. Wash slides 3X in 1X PBS-T (0.3% Triton X-100). 3. Block slides in 1X PBS-T with 3% normal goat serum for 1 hr at room temperature. 4. Incubate slides with primary in 1X PBS-T with 3% normal goat serum (rabbit anti-substance P, ImmunoStar #20064) overnight at 4 °C. We store the primary antibody is stored at a 1:10 dilution and used it in a concentration of 1:4,000. DAY 2 5. Remove antiserum and wash tissue sections 3X 5 min with 1X PBS-T. 6. Incubate slides with secondary antibody (e.g. goat anti-rabbit-Alexa488, Life Technologies, diluted 1:500 in PBS-T) for 2 hrs in a wet-chamber covered by tissue to prevent light bleaching of the secondary antibodies. 7. Wash slides 3X 5 min with PBS-T. 8. Mount with Fluoromount (Southern Biotech) and coverslip. Use fluorescence microscope for visual analysis or imaging.

For our manuscript, Differential roles of ventral pallidum subregions during cocaine self-administration behaviors, in press at Journal of Comparative Neurology, we used the Immunostar rabbit anti-substance P primary antibody to visualize the entire ventral pallidum. Rats were perfused with saline followed by 4% PF, stored in 30% sucrose, and coronally sectioned to 40 um. We followed the protocols of Zahm/Heimer and colleagues when they discovered the ventral pallidum subregions and their afferent/efferent projection patterns. The protocol consisted of the following steps: 1. Wash in 0.1M phosphate buffer (PB; pH 7.4) 2. 15 min in 1% sodium borohydride 3. Wash 4. 1 hour blocking with PB containing 0.1% Triton X-100 and 3% normal goat serum 5. Primary antibody overnight at 4C - ImmunoStar rabbit anti-substance P diluted 1 : 6500 in PB containing 0.1% Triton X-100 and 3% normal goal serum 6. Wash 7. 1 hour anti-rabbit biotinylated secondary (Vector) 1 : 200 in PB with 0.1% Triton X-100 8. Wash 9. 1 hour ABC (Vector) 1 : 200 in PB with 0.1% Triton X-100 10. Wash 11. Develop with 0.05% DAB for 6 min Our study involved recording neurons within the ventral pallidum subregions during specific aspects of intravenous cocaine self-administration (approaching toward, responding on, or retreating away from a cocaine-reinforced operandum). In order to verify the placement of microwires within the VP subregions, prior to perfusion we passed current through each stainless steel microwire to leave an iron deposit at the uninsulated tip. After DAB (brown reaction) and mounting, we visualized the iron deposit by incubating in a 5% potassium ferrocyanide and 10% HCl solution (leaving a blue-green reaction). For our purposes, this antibody strongly labeled fibers in VP with low background similar to what Haber and Nauta 1983 reported.

Tissue: Maccaca fasciscularis, perfused with saline and 4% paraformaldehyde, dehydrated through increase sucrose gradients, sectioned at 40 um. Method: Immunocytochemistry on free floating sections. Incubated with ImmunoStar's primary antibody at a concentration of 1:5000 for 4 days in 10% normal goat serum (in 0.1M PO4 buffer with .3% Triton X-100). Rinsed, then incubated with Vector Lab's biotinylated goat anti-rabbit secondary antibody (#BA-1000) at a concentration of 1:200 for 40 minutes at room temperature in the same 10% normal goat serum solution. Rinsed, then incubated with Vector's standard peroxidase kit (#PK-4000) for 60 minutes at room temperature in 0.1M PO4 buffer with 0.3% Triton X-100. Rinsed, then developed using the instructions for the kit. Results: Low background. Strong staining of fibers, especially in the pallidum. Clear fiber morphology.