Description
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
Host: Rabbit
Quantity / Volume: 100 µL
State: Lyophilized Whole Serum
Reacts With: Bird, Bullfrog, Cat, Chick, Chicken, Dog, Dolphin, Doves, Equine, Fish, Frog, Gerbil, Goat, Goldfish, Guinea Pig, Hamster, Horse, Human, Monkey, Mouse, Mudpuppy, Narwhal, Parrot, Pig, Pig, Pigeon, Quail, Rabbit, Ram (Sheep), Rana Pipiens (Leopard Frog), Rat, Ratfish, Salmon, Scorpaena Porcus (Fish), Sea Lamprey, Sheep, Snail, Sparus Aurata (Fish), Squalus Acanthias (Dogfish), Squirrel, Starfish, Sting Ray, Sturgeon (Fish), Three-Spined Stickleback (Fish), Trout, Turtle, Vole, Zebra Finch, Zebrafish
Availability: In Stock
Alternate Names: Protachykinin-1; PPT; tachykinin peptide hormone; NKA, NKNA, TAC2; tachykinin, precursor 1, anti-substance P
Gene Symbol: TAC1
RRID: AB_572266
Database Links:
Entrez Gene: 100227778 Bird
Entrez Gene: 101095481 Cat
Entrez Gene: 420573 Chick
Entrez Gene: 475239 Dog
Entrez Gene: 101331268 Dolphin
Entrez Gene: 102088620 Doves
Entrez Gene: 100052324 Equine
Entrez Gene: 779508 Frog
Entrez Gene: 102191397 Goat
Entrez Gene: 100713032 Guinea Pig
Entrez Gene: 100760914 Hamster
Entrez Gene: 6863 Human
Entrez Gene: 697350 Monkey
Entrez Gene: 21333 Mouse
Entrez Gene: 104412817 Parrot
Entrez Gene: 100525179 Pig
Entrez Gene: 107309132 Quail
Entrez Gene: 100009275 Rabbit
Entrez Gene: 100034669 Ram
Entrez Gene: 24806 Rat
Entrez Gene: 101972401 Squirrel
Entrez Gene: 102459998 Turtle
Entrez Gene: 101979426 Vole
Entrez Gene: 555634 Zebrafish
Technical Sheets
This product contains the preservative sodium azide. The concentration percent of the sodium azide is ≤ .09%. Although this hazardous substance is a concentration below that required for the preparation of a Material Safety Data Sheet, we created a standard MSDS for your records.
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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
