The antibody is provided as 100 uL of affinity purified serum in PBS (0.02 M sodium phosphate with 0.15 M sodium chloride, pH 7.5) with 1% BSA (bovine serum albumin), and 0.02% sodium azide. Reacts with mouse, rat, and rabbit species.
The images above labeled "ImmunoStar Rabbit 5-HT 2A receptor antibody" are the results of staining of the mouse 5-HT 2A receptor in the brain, courtesy of Dr. Magdalena Zaniewska, Max-Delbruck-Centrum fur Molekulare Medizin Berlin, Germany.
The ImmunoStar 5HT 2A receptor antibody was quality control tested using standard immunohistochemical methods. The antiserum demonstrates strongly positive labeling of rat cortex, amygdala and hippocampus using indirect immunofluorescent and biotin/avidin-HRP techniques. Recommended primary dilutions are 1/300 - 1/500 in PBS/0.3% Triton X-100 - Bn/Av-HRP Technique . The addition of intensifying reagents such as nickel ammonium sulfate to the chromogen solution will approximately double the dilution factor as recommended.
Immunolabeling is completely abolished by preadsorption with synthetic rat 5HT2A receptor (22-41). Immunolabeling of Western blot revealed a single band of approximately 53kD.
Photo Description: Low magnification IHC image of neurons staining for the 5-HT2A receptor in the rat cortex (top of page) and image of neuronal expression of the receptor in the amygdala (below). The bottom right photo is of the cortex. 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 1:500 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 uL
Reacts With: Human, Marine, Mouse, Rabbit, Rat
Alternate Names: 5-hydroxytryptamine receptor 2A; 5Ht-2; 5-hydroxytryptamine (serotonin) receptor 2A, G protein-coupled, anti-5-HT 2A
Gene Symbol / ID, Accession #: Htr2a,29595
03/11/2014 - 10:36:22 AM
Only your AB shows a specific signal
We have been interested in detecting changes of 5-HT2a in rodent brain, primarily by immunoblotting, also by immunohistochemistry. We have tested several commercial antibodies, with only your Ab showing a specific signal, as verified by brain distribution, preadsorbtion and by a lack of signal in KO animals.
We have optimized a immunoblotting protocol which works well to detect 5-HT2a in rat brain lysates. Mouse tissue is a little bit more difficult but still possible
Immunoblotting: Rat or mouse brain samples were lysed by brief sonication in a buffer containing: 1% SDS in 1xPBS, protease inhibitors (Complete Mini, Roche). Samples were mixed with a modified Leamli buffer (containing a reducing agent; 40mM DTT), incubated at 37C for 30mins and separated by SDS-PAGE. Separated proteins were then blotted onto a PVDF membrane, blocked with 5% milk/TBS/0.1% Tween80 and probed with anti-5HT2a antibody (1:500, Immunostar #24288) followed by anti-rabbit HRP- conjugated secondary antibody (1:5000, Jackson Immuno). Signal was visualized using ECL2 kit according to manufacturer's instructions (Thermo).
Image Samples: 1 (Rat prefrontal cortex, 25ug), 2 (Rat laterodorsal thalamus, 25ug), 3 (Wt mouse cortex, 25 ug), 4 (5-HT2a KO mouse cortex, 25 ug), 5 (Wt mouse cortex, 75 ug), 6 (5-HT2a KO mouse cortex, 75 ug). As expected, sample in lane 2 showed low expression, and samples in lanes 4 and 6 showed no expression of 5-HT2a receptors. Mouse tissues obtained from Dr. Gonzalez-Maeso (Mount Sinai Hospital, NY) indicates detected 5Ht2a receptor signal.
Marek Schwendt, PhD
University of Florida
11/15/2012 - 02:37:02 PM
Antibodies are specific for mouse receptor
Here are the results of staining of the mouse 5-HT 2A receptors in the brain with the use of Immunostar's antibodies (Cat no: 24288). I think the antibodies are specific for the mouse receptor (no receptor labeling in the brain of 5-HT 2A receptor knockout mouse) and can be used in a Western blot. What I already found out is that 5-HT 2A receptors in the cortex seem to be glycosylated (band around 75 kDa) since incubation with PNGaseF releases the protein that is around 50 kDa.
We received the 5-HT 2A receptor knockout animals from Dr. Joelle Adrien from another research team in Paris (see below).
Dr. Magdalena Zaniewska
Molekularbiologie von Hormonen im Herz-Kreislaufssystem
Max-Delbrϋck-Centrum fϋr Molekulare Medizin
Dr. Joelle Adrien
Directeur de Recherches a l'Inserm
Faculte de Medecine Pierre et Marie Curie - Site Pitie Salpetriere
|PROD #||ANTIBODY||CITATION||SPECIES REACTIVITY||APPL METHOD||RESEARCH AREA||# CITED BY||PUBMED ID|
|24288||5-HT (Serotonin) 2A Receptor Antibody||Fay, Richard, and Leszek Kubin. "5-HT 2A receptor-like protein is present in small neurons located in rat mesopontine cholinergic nuclei, but absent from cholinergic neurons." Neuroscience letters 314.1 (2001): 77-81.||Rat||19||11698151|
|24288||5-HT (Serotonin) 2A Receptor Antibody||Vis Neurosci. 1999 Mar-Apr;16(2):221-30. 5-HT2a receptors in the rabbit retina: potential presynaptic modulators. Pootanakit K1, Prior KJ, Hunter DD, Brunken WJ.||Rabbit||IHC||10367957|
|24288||5-HT (Serotonin) 2A Receptor Antibody||Coulon P1, Kanyshkova T, Broicher T, Munsch T, Wettschureck N, Seidenbecher T, Meuth SG, Offermanns S, Pape HC, Budde T, Front Cell Neurosci. 2010 Oct 25;4:132. doi: 10.3389/ fncel.2010.00132. eCollection 2010. "Activity Modes in Thalamocortical Relay Neurons are Modulated by G(q)/G(11) Family G-proteins - Serotonergic and Glutamatergic Signaling"||Mouse||IF||21267426|
|24288||5-HT (Serotonin) 2A Receptor Antibody||Kwan, Marilyn L., et al. "Airway vasculature after mycoplasma infection: chronic leakiness and selective hypersensitivity to substance P." American Journal of Physiology-Lung Cellular and Molecular Physiology 280.2 (2001): L286-L297.||Rat||IHC||24||11159008|
|24288||5-HT (Serotonin) 2A Receptor Antibody||Vázquez DM1, Eskandari R, Zimmer CA, Levine S, López JF, Psychoneuroendocrinology. 2002 Jan-Feb;27(1-2):245-72. "Brain 5-HT receptor system in the stressed infant rat: implications for vulnerability to substance abuse"||Rat||ICC||56||11750782|
|24288||5-HT (Serotonin) 2A Receptor Antibody||Itoh, Takeo, and Junko Kajikuri. "Characteristics of the actions by which 5?HT affects electrical and mechanical activities in rabbit jugular vein." British journal of pharmacology 164.3 (2011): 979-991.||Rabbit||IF||2||21449974|
|24288||5-HT (Serotonin) 2A Receptor Antibody||Maekawa, Takashi, et al. "Characteristics of the actions by which 5?hydroxytryptamine affects electrical and mechanical activities in rabbit jugular vein graft." British journal of pharmacology 166.4 (2012): 1419-1432.||Rabbit||IF||4||22251164|
|24288||5-HT (Serotonin) 2A Receptor Antibody||Rasul, Aram, et al. "Chronic mild stress modulates 5-HT1A and 5-HT2A receptor expression in the cerebellar cortex of NC/Nga atopic-like mice." Archives of dermatological research 305.5 (2013): 407-413.||Mouse||IHC||0||23440396|
|24288||5-HT (Serotonin) 2A Receptor Antibody||J Vasc Surg. 2009 Sep;50(3):617-25. doi: 10.1016/j.jvs.2009.05.065. Chronic treatment of hydroxytryptamine type 2a receptor antagonist sarpogrelate hydrochloride modulates the vasoreactivity of serotonin in experimental rabbit vein grafts. Kodama A1, Komori K, Kajikuri J, Itoh T.||Rabbit||IF||19700096|
|24288||5-HT (Serotonin) 2A Receptor Antibody||Volgin, Denys V., Georg M. Stettner, and Leszek Kubin. "Circadian dependence of receptors that mediate wake-related excitatory drive to hypoglossal motoneurons." Respiratory physiology & neurobiology 188.3 (2013): 301-307.||Rat||0||23665050|
|24288||5-HT (Serotonin) 2A Receptor Antibody||Pearlstein, E., et al. "Contribution of 5?HT to locomotion||Mouse||IHC||5||21501257|
|24288||5-HT (Serotonin) 2A Receptor Antibody||Zeng, Fanqing, Chen Xu, and Ge Xu. "Distribution of serotonin 5-HT2A and 5-HT7 receptors in the Onuf's nucleus of the rat spinal cord." Neural Regen Res 3.8 (2008): 853-857.||Rat||2||not found|
|24288||5-HT (Serotonin) 2A Receptor Antibody||Rasul, Aram, et al. "Effect of chronic mild stress on serotonergic markers in the skin and brain of the NC/Nga atopic-like mouse strain." Archives of dermatological research 303.9 (2011): 625-633.||Mouse||9||21400247|
|24288||5-HT (Serotonin) 2A Receptor Antibody||Br J Pharmacol. 2010 Jul;160(5):1092-104. doi: 10.1111/j.1476-5381.2010.00753.x. Enhancement of mesenteric artery contraction to 5-HT depends on Rho kinase and Src kinase pathways in the ob/ob mouse model of type 2 diabetes. Matsumoto T1, Kobayashi T, Ishida K, Taguchi K, Kamata K.||Mouse||WB||20590603|
|24288||5-HT (Serotonin) 2A Receptor Antibody||Exp Neurol. 2013 Sep;247:572-81. doi: 10.1016/j.expneurol.2013.02.008. Epub 2013 Feb 26.Grafting of fetal brainstem 5-HT neurons into the sublesional spinal cord of paraplegic rats restores coordinated hindlimb locomotion.Sławińska U1, Miazga K, Cabaj AM, Leszczyńska AN, Majczyński H, Nagy JI, Jordan LM.||Rat||1||23481546|
|24288||5-HT (Serotonin) 2A Receptor Antibody||Weber, Elaine T., and Rodrigo Andrade. "Htr2a gene and 5-HT2A receptor expression in the cerebral cortex studied using genetically modified mice." Frontiers in neuroscience 4 (2010).||Mouse||31||20802802|
|24288||5-HT (Serotonin) 2A Receptor Antibody||Hayashi, Takayuki, et al. "Intracerebroventricular fluvoxamine administration inhibited pain behavior but increased Fos expression in affective pain pathways." Pharmacology Biochemistry and Behavior 91.3 (2009): 441-446.||IHC||6||18817806|
|24288||5-HT (Serotonin) 2A Receptor Antibody||Stettner, Georg M., Leszek Kubin, and Denys V. Volgin. "Loss of motoneurons in the ventral compartment of the rat hypoglossal nucleus following early postnatal exposure to alcohol." Journal of chemical neuroanatomy 52 (2013): 87-94.||Rat||0||23932955|
|24288||5-HT (Serotonin) 2A Receptor Antibody||Fay, Richard, and Leszek Kubin. "Pontomedullary distribution of 5?HT2A receptor?like protein in the rat." Journal of Comparative Neurology 418.3 (2000): 323-345.||Rat||96||10701830|
|24288||5-HT (Serotonin) 2A Receptor Antibody||Bras H1, Gaytán SP, Portalier P, Zanella S, Pásaro R, Coulon P, Hilaire G, Eur J Neurosci. 2008 Sep;28(6):1097-107. doi:0.1111/j.1460- 9568.2008.06407.x. Epub 2008 Sep 9. "Prenatal activation of 5-HT2A receptor induces expression of 5-HT1B receptor in phrenic motoneurons and alters the organization of their premotor network in newborn mice".||Mouse||ICC||12||18783379|
|24288||5-HT (Serotonin) 2A Receptor Antibody||Karaki, Samah, et al. "Quantitative Phosphoproteomics Unravels Biased Phosphorylation of Serotonin 5-HT2A Receptor at Ser280 by Hallucinogenic versus non-Hallucinogenic Agonists." Molecular & Cellular Proteomics (2014): mcp-M113.||0||not found|
|24288||5-HT (Serotonin) 2A Receptor Antibody||Kong, Xiang-Yu, et al. "Robust upregulation of serotonin 2A receptors after chronic spinal transection of rats: An immunohistochemical study." Brain research 1320 (2010): 60-68.||Rat||IF||13||20085755|
|24288||5-HT (Serotonin) 2A Receptor Antibody||Van Steenwinckel, Juliette, et al. "Role of spinal serotonin 5-HT2A receptor in 2?, 3?-dideoxycytidine-induced neuropathic pain in the rat and the mouse." Pain 137.1 (2008): 66-80.||Rat, Mouse||27||17888573|
|24288||5-HT (Serotonin) 2A Receptor Antibody||Rasul, Aram. "Serotonergic mechanisms in atopic dermatitis." (2012).||0||not found|
|24288||5-HT (Serotonin) 2A Receptor Antibody||Xu, Chen, et al. "Serotonin 5?HT2A and 5?HT5A receptors are expressed by different motoneuron populations in rat Onuf's nucleus." Journal of Comparative Neurology 502.4 (2007): 620-634.||Rat||IHC||23||17394137|
|24288||5-HT (Serotonin) 2A Receptor Antibody||Thibault, Karine, et al. "Serotonin 5-HT2A receptor involvement and Fos expression at the spinal level in vincristine-induced neuropathy in the rat." Pain 140.2 (2008): 305-322.||Rat||32||18930597|
|24288||5-HT (Serotonin) 2A Receptor Antibody||Loyd, Dayna R., et al. "Sex differences in serotonin enhancement of capsaicin-evoked calcitonin gene-related peptide release from human dental pulp." Pain 153.10 (2012): 2061-2067.||Human||6||22819536|
|24288||5-HT (Serotonin) 2A Receptor Antibody||Neuropsychopharmacology. 2009 Jan;34(2):410-23. doi: 10.1038/npp.2008.71. Epub 2008 Jun 4. Stress impairs 5-HT2A receptor-mediated serotonergic facilitation of GABA release in juvenile rat basolateral amygdala. Jiang X1, Xing G, Yang C, Verma A, Zhang L, Li H.||Rat||IF||18536707|
|24288||5-HT (Serotonin) 2A Receptor Antibody||Dumitrascu, R., et al. "Terguride ameliorates monocrotaline-induced pulmonary hypertension in rats." European Respiratory Journal 37.5 (2011): 1104-1118.||Rat||IHC||44||20947677|
|24288||5-HT (Serotonin) 2A Receptor Antibody||Van Steenwinckel, J., et al. "The 5-HT2A receptor is mainly expressed in nociceptive sensory neurons in rat lumbar dorsal root ganglia." Neuroscience 161.3 (2009): 838-846.||Rat||IHC||27||19362128|
|24288||5-HT (Serotonin) 2A Receptor Antibody||J Comp Neurol. 2004 May 10;472(4):496-511. The 5-HT2A receptor is widely distributed in the rat spinal cord and mainly localized at the plasma membrane of postsynaptic neurons. Doly S1, Madeira A, Fischer J, Brisorgueil MJ, Daval G, Bernard R, Vergé D, Conrath M.||Rat||ICC||68||15065122|
|24288||5-HT (Serotonin) 2A Receptor Antibody||Neurochem Res. 2005 un-Jul;30(6-7):825-37. The pre-nervous serotonergic system of developing sea urchin embryos and larvae: pharmacologic and immunocytochemical evidence.Buznikov GA1, Peterson RE, Nikitina LA, Bezuglov VV, Lauder JM.||Marine||IHC||16187217|
|24288||5-HT (Serotonin) 2A Receptor Antibody||Kong, X-Y., et al. "The time course of serotonin 2A receptor expression after spinal transection of rats: an immunohistochemical study." Neuroscience 177 (2011): 114-126.||Rat||9||21211552|
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Q & A Wall
I would be very grateful if you could send me the Western Blot protocol for 5-HT2A R Ab. Did you prepare the WB from the brain tissue homogenate?
Question By Admin
Western blots of 5HTRs is challenging. We have examined crude homogenates and immunopurified tissue extracts. The extraction buffer was RIPA buffer with enzyme inhibitor tablets added (Sigma). We used the Pierce Seize X mammalian immunoprecipitation kit 45210.
Due to the difficulty in running westerns with this antibody, ImmunoStar does not support or warrant the western blot application. The western information is included as specificity information only.
For those that wish to proceed with the western blot, here is a summary of our protocol for the 5-HT2A antibody:
1. Tissue is homogenized in either electrophoresis sample bufferor (in which case you go straight to electrophoresis with no cleanup) or in 0.05 Mis-saline with 10% sucrose and 1 mM PMSF, pH 7.6 (in which case you proceed as below).
2. Centrifuge homogenized sample and collect pellet by centrifugation. Solubilize pellet in 20 mM tris-saline, pH 8, 1 mM EDTA, and 1% deoxycholate, sonicate and incubate hr on ice. Centrifuge and collect supernatant. Subject supernatant to electrophoresis.
3. Electrophorese and transfer onto nitrocellulose by standard methods.
4. Block membrane in blotto buffer (TBS with 0.05% Tween 20 and 0.2% I-block [Tropix #AI300]. This is also the antibody incubation buffer.
5. Incubate blots in rab anti 5HT2a at 1:500 overnight in refrigerator.
6. Proceed with chemiluminescent detection (Amersham ECL kit)
Answer By Admin on 2015-06-11 04:37:52
We would like to know if this antibody cross-reacts with human. We want to use it on sections for immunohistochemistry.
Question By Admin
Our antibody was raised against rat 22-41. This sequence blasted and got 78% similarity to human, though not a lot of amino acids in a row to say it will definitely x-react. We don't know if anyone has used in human and published either.
Here's the alignment:
Answer By Admin on 2015-06-09 06:20:52
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.Download MSDS