The ImmunoStar Substance P antiserum was quality control tested using standard immunohistochemical methods. The antiserum demonstrates strongly positive labeling of rat substantia nigra and spinal cord using biotin/avidin-HRP techniques. Recommended primary dilution is 1/6000-1/8000 in PBS/0.3% Triton X-100 – biotin/avidin-HRP Technique.
The specificity of the antiserum for Substance P was demonstrated using soluble pre-adsorption with the peptides in question at a final concentration of 10 µg of peptide per mL of diluted antiserum. Substance P immunolabeling was completely abolished by pre-adsorption with Substance P. Pre-adsorption with the following peptides resulted in no reduction of immunostaining: neurokinin A, neurokinin B, somatostatin and neuropeptide K.
Photo Description: IHC image of rat spinal cord (top above) and rat tegmentum (below) staining for substance p. The tissue was fixed with 4% formaldehyde in phosphate buffer, before being removed and prepared for vibratome sectioning for brain and frozen sectioning for spinal cord. Sections were incubated at RT in 10% goat serum in PBS, before standard IHC procedure. Primary antibody was incubated at 1:5000 for 48 hours, goat anti-rabbit secondary was subsequently added for 1 hour after washing with PBS. Light microscopy staining was achieved with standard biotin-streptavidin/HRP procedure and DAB chromogen.
The immunofluorescent photo (second from the top)is wholemount mouse duodenum immunostained for Substance P (green, Alexa Fluor 488) and ANNA (neuron marker; red, Alexa Fluor 647). Photo courtesy of Ryan Hamnett, PhD, Department of Neurosurgery Stanford University
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Mouse enteric neuron staining in whole-mount tissue
We successfully stained substance P neurons in the mouse myenteric plexus.
Mouse small intestine was removed and the longitudinal muscle and myenteric plexus (LMMP) dissected out in cold PBS. Tissue was pinned and fixed in 4% PFA at 4C for 90 mins, then washed 3 times in PBS (10 mins each).
IHC: 1. Tissue is cut into small pieces and transferred to wells containing PBS for staining. 2. Tissue is incubated overnight at 4C (shaking) in a 1:2000 dilution of rabbit anti-substance P in PBT (PBS, 1% BSA, 0.1% triton X-100). 3. The following day, 3×10 min washes in PBT at room temperature 4. 2 h incubation in Alexa 488 donkey anti-rabbit, room temperature, shaking. 5. 2×10 min washes in PBT, 2×10 min washes in PBS 6. Tissue is transferred onto a slide and allowed to dry. 7. Once dry, slide is rinsed in water. 8. Slide is coverslipped with appropriate mounting medium (in this case, Prolong Glass). 9. Confocal imaging performed on Leica SP8.
Ryan Hamnett, PhD Department of Neurosurgery Stanford University
Strong reactivity in crayfish brain
Tissue: Procambarus clarkii, perfused with saline and 4% paraformaldehyde, Brains were embedded in gelatin and sectioned with a vibratome to 60 um.
Protocol: Floating section immunohistochemistry methods followed that of Lin and Strausfeld, 2013.
1.permeabilize tissue with 0.5% PBS-TX 2.wash 3.block in 0.5% PBS-TX + 5% normal goat serum 4.incubate on shaker overnight with Immunostar rabbit anti-Substance P (1:200 in -.5% PBS-TX + 5% NGS) 5.wash 6.incubate on shaker overnight with Life Technologies goat anti-rabbit-Alexa488 (1:400 in 0.5% PBS-TX + 5% NGS) 7.wash 8.mount with Elvanol and coverslip
Results: Clear staining of fibers throughout the brain.
Alice Chou, PhD Candidate Department of Biology University of Maryland Baltimore County
Success With Adult Zebrafish Brain
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.
Thomas Mueller (Ph.D.) Division of Biology Kansas State University
Ventral pallidum visualization
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.
David H. Root, Ph.D. Rutgers University
Strong Staining of Fibers
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.
Daniel Tylee Department of Neurobiology & Anatomy University of Rochester – School of Medicine and Dentistry