The ImmunoStar serotonin antiserum was quality control tested using standard immunohistochemical methods. The antiserum demonstrates strongly positive labeling of rat hypothalamus, raphe nuclei and spinal cord using indirect immunofluorescent and biotin/avidin-HRP techniques. Recommended primary dilution is 1/20,000-1/40,000 in PBS/0.3% Triton X-100 – biotin/avidin-HRP Technique. Staining is completely eliminated by pretreatment of the diluted antibody with 25 ug of serotonin/BSA.
Cross reactivity of Serotonin antisera was examined. With 5µg, 10µg and 25µg amounts the following substances did not react with Serotonin antisera diluted 1/20,000 using the Bn-SA/HRP labeling method: 5-hydroxytryptophan, 5-hydroxyindole -3- acetic acid, and dopamine.
Photo Description: Low magnification IHC image of neurons staining for the 5-HT rabbit antibody in the raphe nucleus of the rat brainstem (above), and corresponding high magnification image of the raphe nucleus (below). The tissue was fixed with 4% formaldehyde in 0.1 M phosphate buffer, before being removed and prepared for vibratome sectioning. Floating sections were incubated at RT in 10% goat serum in PBS, before standard IHC procedure. Primary antibody was incubated at 1:25000 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.
Quantity / Volume: 100 µL
State: Lyophilized Whole Serum
Availability: In Stock
Reacts With: Abalone (Haliotis Kamtschatkana) , Amphibian, Annelid Mushroom, Annelida (Segmented Worm), Ant, Ant (Pheidole Dentata), Antalis Entalis Scaphopoda, Aplysia (Sea Slug), Aplysia Californica (Sea Slug), Aplysia Punctata (Sea Slug), Arachnocampa (Gnat), Archidoris Pseudoargus (Sea Slug), Asterina Pectinifera (Starfish ), Astyanax Mexicanus (Cave Fish), Axolotl (Salamander), Bacteria, Beetle, Bird, Brachiopods, Bryozoans (Polyzoa), Buffalo, Bufo Marinus (Toad), Bug, Bumblebee, Butterfly, Butterfly (Monarch), Canine, Cat, Chick, Chicken, Chiton, Chrysoperla Carnea Green Lacewing , Clam, Clione Limacina Pteropod (Fruit Bat), Cockroach, Cod, Crab, Crayfish, Cricket, Crustaceans, Cupiennius Salei (Spider), Desert Locust, Dog, Dogfish (Shark), Dove, Drosophila (Fly), Drosophila Melanogaster (Fly), Feather Star, Ferret, Fish, Fish (Salmon), Flounder, Fly, Frog, Frog (Xenopus Laevis), Fruit Fly, Gastropod (Slug), Gastropod (Slug), Gerbil, Glowworm, Goldfish, Grasshopper, Guinea Pig, Hamster, Hedgehog, Helisoma Trivolvis (Snail), Hen, Hermit Crab, Honeybee, Human, Idiosepius Notoides (Squid), Ilyanassa Obsoleta (Sea Snail), Insect, Kitten (Cat), Lamprey, Leech, Lizard, Lobster, Lobster (Cycliophora), Lobster Larva, Locust, Lythrypnus Dalli (Fish), Macracantha (Cephalocarida), Manduca Sexta (Moth), Marmoset (Monkey), Mercenaria (Clam), Mexican salamander (Ambystoma Mexicanum) , Mollusca, Mollusk, Monkey, Monkey (Macaque), Mosquito, Mosquito (Aedes Aegypti), Moth, Moth (Manduca Sexta), Mouse, Mudpuppy, Mussel, Newt, Nudibanch, Nudibranch Mollusc, Nudipleura, Octopus, Onychophora (Velvet Worm), Ovine (Sheep), Parhedyle Cryptophthalma (Sea Slug), Phoronis Pallida (Phoronida), Phyllodoce Maculata (Phyllodocidae), Phyllodoce Maculata (Polychaeta), Pig, Pigeon, Pilidium Larvae, Platynereis Dumerilii (Ragworm), Platypus, Pleurobranchaea Californica (Sea Slug), Pleurobranchaea Japonica (Sea Slug), Pleurodeles (Newt), Prawn, Praying Mantis, Ptychodera Flava (Worm), Quail, Rabbit, Raja Erinacea (Skate), Ram (Sheep), Rana Perezi (Frog), Rat, Red Abalone, Red Abalone, Renilla Koellikeri (Cnidarian ), Rhodnius Prolixus (Bug), Rhodnius Prolixus (Parasite), Salamander, Sand Dollar, Schistocerca Gregaria (Locust), Scorpaena Porcus (Fish), Scorpion, Scyliorhinus Canicula (Dogshark), Sea Bass (Fish), Schistocerca Gregaria (Locust), Scorpaena Porcus (Fish), Scorpion, Scyliorhinus Canicula (Dogshark), Sea Bass (Fish), Sea Cucumber, Sea Lamprey, Sea Lily, Sea Scallop, Sea Slug, Sea Spider, Sea Star, Sea Urchin, Seahorse, Shark, Sheep, Shrew, Skate, Snail, Sparus Aurata (Fish), Spisula (Clam), Springtails, Squirrel Monkey, Sting Ray, Stomatopod Crustacean, Sturgon, Tapeworm, Tectura Scutum (Mollusk), Tick, Tigriopus Californicus (Copepod), Toad, Tragulus Javanicus (Mouse Deer), Tritonia Diomedea (Sea Slug), Trout, Turkey, Turtle, Worm, Worm (Digenea) , Worm (Diphyllobothrium Dentriticum) , Worm (Dugesia Tigrina) , Worm (Dugesia Tigrina), Worm (Phyllodoce Groenlandica), Worm (Polychaete Capitella), Zebra Finch (Bird), Zebrafish
Alternate Names: anti-5-HT Rabbit
Gene Symbol: Spl
Entrez Gene ID: 104153
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I have used this antibody to stain for 5HT positive cells in rat spinal cord after rehabilitation or transplantation therapies. I have always gotten consistent and beautiful staining at dilutions 1:80000 for DAB and 1:20000 for IF.
Tissue preparation for rat tissue:
TTranscardial perfusion using 4% paraformaldehyde, post fixed overnight in 4% para, and cryoprotected in 30% sucrose for 3 days. Freeze tissue, sectioned (at 40 micron), and use immediately within days.
Indirect Immunofluorescence staining protocol:
1. wash 1x PBST for 15 min
2. blocking solution (1x PBS + 0.3% Triton X100, 3% goat serum) for 1 hr at RT
3. Incubate tissue sections to primary antiserum (rabbit anti-5HT, ImmunoStar #20080) diluted in blocking buffer overnight @ 4′;C.
5. wash tissue sections 3 x 10 min with 1xPBS.
6. Incubate tissue sections with secondary antibody (e.g. goat anti-rabbit-Alexa Fl 488, Jackson ImmunoResearch Laboratories, Inc., diluted 1:500 in PBS) for 1-2 hrs in the dark at room temperature.
7. wash tissue sections 3 x 15 min with 1x PBS in the dark.
8. Coverslip with vectashield and store at RT.
Love the Consistency and Reliability
I have used the 5-HT Rabbit antibody (# 20080) for both my honors and masters project in multiple species including sea urchin (Lytechinus variegatus), polychaete (Capitella telata), and two cephalopods (Sepia officinalis and Loligo pealeii).
I use my antibodies in immunohistochemistry in whole mount preparations in 1:200 dilutions.
This antibody reliably stains the cell bodies and axons of the central nervous system in all my invertebrate preps. I love the consistency of staining I get across my study specimen, and the reliability of the product.
Student of Dr. Roger Croll
We have used this antibody to stain for 5HT positive cells in tissue sections (rat brain stem and spinal cords tissue samples) or in cultured mouse ventral mesencephalic cells. We have always gotten consistent and beautiful staining at dilutions 1:1000 – 1:5000.
Tissue preparation for rat tissue:
TTranscardial perfusion with 4% paraformaldehyde in 0.1M sodium phosphate buffer (PBS), pH 7.4, post fixed overnight in 4% para overnight, and cryoprotected in 30% sucrose until they sink. Freeze tissue, sectioned (at 16 micron), and thaw mounted on to gelatin coated slides. Samples are stored in -80;C until use.
Indirect Immunofluorescence staining protocol:
1. Remove slides from -80’C freezer and equilibrate to room temperature for at least 2 hrs.
2. Immerse slides in 1x PBST for 15 min
3. Remove slides from PBST and expose tissue sections to blocking solution (1x PBS + 0.3% Triton X100, 3% goat serum) for 1 hr at RT.
4.Incubate tissue sections to primary antiserum (rabbit anti-5HT, ImmunoStar #20080) diluted in blocking buffer overnight @ 4′;C.
5. Wick off antiserum and wash tissue sections 3 x 10 min with 1xPBS.
6. Incubate tissue sections with secondary antibody (e.g. goat anti-rabbit-Alexa Fl 488, Jackson ImmunoResearch Laboratories, Inc., diluted 1:500 in PBS) for 1-2 hrs in the dark at room temperature. After incubation with secondary antibody, wash tissue sections 3 x 15 min with 1x PBS in the dark.
7. Counterstain with DAPI.
8. Coverslip with glycerol gel and store at RT.
For in vitro cultured cells:
1. Fix samples with 2% paraformaldehyde for 20 min at room temperature.
Follow the exact protocol for staining.
Zin Khaing, PhD
Research Assistant Scientist
Department of Biomedical Engineering
BMS Building, J298
The University of Florida
Very Reliable Antibody
I have used this antibody (#20080) to label the brains of several dozen different species of insects. Brains are embedded in low melting point agarose and sectioned. I have obtained bright labeling with dilutions as low as 1:80,000, although I typically use 1:4,000. Pre-adsorbing the antibody with BSA did not affect labeling of brain tissue of the moth Manduca sexta (see Dacks et al 2006 in J. Comp. Neurol.) and pre-adsorbing the antibody with 40mg/ml 5HT-BSA conjugate (immunostar cat# 20081) resulted in no labeling at a 1:20,000 primary antibody dilution (see Paulk et al 2009, J. Comp. Neurol.).
______Dissect and Fix tissue overnight in 4% paraformaldehyde @ 4oC.
______Wash 3X5min in phosphate buffered saline (PBS; pH 6.9)
______Embed and section at 100um in PBS with 0.5% Triton X-100 (PBST).
______Wash 4x 1/2Hr PBST at room temperature (RT).
______Block 1hr in 2% IgG-free bovine serum albumin (BSA) in PBST @ RT.
______Incubate in primary (Rb anti-5HT, 1:4000; Immunostar #20080) in 2% (BSA) in PBST with 50mM sodium azide (PBSAT) overnight @ RT .
______Wash 5X 1/2Hr PBST at RT.
______Block as Day 2 for 1 hour
______Spin down Cy3 in PBSAT with 2% BSA
______Incubate in secondary (Gt anti-Rb Cy3, 1:1,000) in 2% BSA in PBSAT overnight @ RT.
______Wash 2X 30 min in PBST
______Wash 2X 30 min in PBS
______Wash 5 min in 60% glycerol
______Mount in 80% glycerol
West Virginia University
We have used this antibody (#20080) recently for several months on water flea Daphnia brains at 1:1000 to 1:2000 dilutions with excellent results in whole mounts and frozen sections. On tissues fixed even in a fixative especially suitable for neuropeptides, the antibody worked surprisingly superb showing clearly 5HT-labelled somata, fibres and finest terminals that were easily detected also in double-stained preparations with other antibodies.
All procedures were performed at room temperature; no cold incubations in 4°C environments were necessary.
Brains were fully or partially dissected in physiological saline and fixed either in 4% paraformaldehyde (pFA) in 0.1M sodium phosphate buffer (PBS), pH 7.4 or Stefanini’s fixative containing picric acid (Stefanini et al. Nature 216:173-174, 1967; especially suitable for neuropeptides) 4 hours to overnight at room temperature.
– Whole mounts were rinsed in 0.1M PBS and finally 3-4 times 10min in Tris-buffered saline (0.1M Tris-HCl-buffered saline containing 150mM NaCl and 0.5% Triton X 100; pH 7.4; TBTX).
– Tissues for cryostat sectioning were rinsed several times in 0.1M PBS and then infiltrated in a gradient of 10%, 20%, 30% sucrose/0.1M PBS at 4°C until they sank and left for ca. 1 hour each, finally embedded in Tissue Tek and frozen. Frozen tissues were sectioned at 15 µm, thawed onto glass slides, and stored at -20°C until staining.
Indirect immunofluorescence staining protocol (all procedures at room temperature):
Slides or whole mounts were washed several times in TBTX; a blocking solution thereafter was found as unnecessary; only whole mounts were gently shaken in solutions.
Primary antiserum (rabbit anti-5HT, ImmunoStar #20080) was applied overnight at room temperature (RT) diluted 1:1000 or 1:2000 in TBTX (0.02% sodium azide was added to prevent evtl. bacterial growth) overnight.
Samples were washed thereafter several times in TBTX.
Incubation in secondary antibodies (e.g. goat anti-rabbit-Cy3, Jackson ImmunoResearch Laboratories, Inc., diluted 1:500 in TBTX, or Sigma F0382 goat anti-rabbit FITC 1:100 in TBTX, containing 0.02% sodium azide) took place for one hour at RT followed by several washings in TBTX.
Finally Triton X 100 was washed out a few times in 0.1M PBS, and whole mounts or sections were mounted in 80% glycerol/ water containing 0.5mg/ml DABCO (Diaza-bicyclo-octane, Fluka, a very nice antifade solution allowing even confocal imaging for many hours (!!) without any signs of fading).
Both fixatives 4% pFA in PBS and Stefanini’s give usually similar results, however, in our opinion the latter picric acid-containing fixative gives more reliable results and at the same time is well suited for peptide-colocalisation studies.
Heinrich Dircksen, PhD
Dept of Zoology
10691 Stockholm, Sweden
We have routinely used this antibody to stain for serotonin in the dorsal raphe of the mouse brain for several years with excellent results.
Mice are perfused with PBS and 4% paraformaldehyde and the brain is removed, post-fixed in 4% paraformaldehyde for 2 hrs and frozen in OCT. Sections are cut using a cryostat and stored at -20 °C until use.
Immunofluorescence staining protocol:
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 5% normal goat serum (or serum of animal host of secondary antibody) for 1 hr at room temperature.
4. Incubate slides with primary in 1X PBS-T with 5% normal goat serum (rabbit anti-5-HT, ImmunoStar #20080) overnight at 4 °C. Primary antibody is stored at a 1:100 dilution and used from 1:1,000 to 1:5,000.
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 dark at room temperature.
7. Wash slides 3X 5 min with PBS-T in the dark.
8. Mount with Prolong gold (Life Technologies), coverslip and visualize on fluorescent microscope or store at -20 °C.
Evan Deneris Lab – Case Western Reserve University
Our lab has used the rabbit antibody against 5HT (#20080) in the rat and monkey spinal cord tissue with phenomenal results. In our hands, we got excellent staining of 5HT antibody diluted at 1:1000 in fibers and terminals with none to very low background staining.
Generally we will perfuse, dissect and fix the tissue for 30min to 1 hour in 4% Paraformaldehyde. After cryopreservation in a 30% sucrose solution (1xPBS), the tissue is frozen in embedding media, cryosectioned, and mounted on microscope slides ready to be stained. Our immunohistochemistry protocol for this antibody is as follows:
1. Post-fix sections in cold Acetone(-20°C) for 10 min at -20°C.
2. Wash slides 3X5 min. in 0.05M Tris Buffered Saline (TBS) pH7.4 .
3. Incubate sections with blocking solution (5% fishskin gelatin, 0.05% Triton X-100 at RT, 0.025 Sodium Azide)for 1 hour at room temperature.
4. Wash slides 2X5 min. in TBS.
5. Incubate in 1:1000 Rb/5HT overnight at 4°C.
1. Wash slides with TBS 3X5min.
2. Incubate slides with secondary antiserum diluted in blocking buffer for 1 hour at room temp (i.e. donkey anti rabbit-Alexa 555 1:1000, Invitrogen).
3. Wash slides 2X5min. in TBS.
4. Wash slides 2X5min in ddWater.
5. Coverslip with Vectashield and view or store at 4°C.
Edgardo J. Arroyo, Ph. D.
VA CT HealthCare System/ Yale University
950 Campbell Ave. Bldg 34
West Haven, CT 06516
I have used this antibody (#20080) for several years on neonatal mouse brainstem, and at a much higher than the 1:1000 to 1:2000 recommended dilution, with excellent results. In adequately fixed tissue, the antibody produces intense staining in 5-HT cell bodies in the raphe nuclei as well as crisp fiber and terminal staining throughout the brainstem.
The mice are perfused transcardially with 4% paraformaldehyde in 0.1M sodium phosphate buffer (PBS), pH 7.4 and the head of the mouse is blocked to remove the brainstem in situ. The tissue is post-fixed overnight at 4°C in 4% paraformaldehyde, then placed in 30% sucrose/0.1M PBS at 4°C until they sink. The tissue is then transferred to a 1:1 mix of 30% sucrose/Tissue Tek for 24 hours (at 4°C) before being embedded in Tissue Tek and frozen over dry ice. Frozen tissue blocks are sectioned at 20 µM, thaw-mounted onto glass slides, and stored at -20°C until use.
Indirect Immunofluorescence staining protocol:
1. Remove slides from -20°C freezer and equilibrate to room temperature
2. Immerse slides in 1x PBST (890 ml of H2O + 100 ml of 10x PBS + 10 ml of 10% Triton X-100) for 15 min
3. Remove slides from PBST and expose tissue sections to blocking solution (1x PBST + 2% sheep serum) for 1 hr at room temperature
4. Expose tissue sections to primary antiserum (rabbit anti-5HT, ImmunoStar #20080) overnight @ 4°C. Primary antibody is stored at a 1:10 dilution and used at 1:8,000
5. Wick off antiserum and wash tissue sections 3 x 10 min with 1xPBST
6. Incubate tissue sections with secondary antibody (e.g. goat anti-rabbit-FITC, Jackson ImmunoResearch Laboratories, Inc., diluted 1:200 in PBST) for 2 hrs in the dark at room temperature. After incubation with secondary antibody, wash tissue sections 3 x 10 min with 1x PBST in the dark
7. Treat tissue sections with freshly made p-phenylenediamine (1 mg/ml) for 1 min, then wash 2 x 10 min in 1x PBS.
8. Coverslip with glycerol gel and store at -20°C
Jeffery T. Erickson, PhD
The College of New Jersey
Ewing, NJ 08628