VR1 N-Terminus (TRPV1)-Rabbit

Marina Quartu,Maria Pina Serra,Marianna Boi,Laura Poddighe, Cristina Picci,Roberto Demontis,and Marina Del Fiacco. TRPV1 receptor in the human trigeminal ganglion and spinal nucleus: immunohistochemical localization and comparison with the neuropeptides CGRP and SP. Journal of Anatomy. July 2016. DOI: 10.1111/joa.12529

... The following commercially available rabbit polyclonal antisera against TRPV1 were tested: Thermo Scientific Pierce-Fisher (TS), raised against a synthetic peptide corresponding to residues T(7)DLGAAADPLQKDTC(21) of the human protein; Neuromics (N), raised against the ...

Krisztina Pohóczky, József Kun, Bálint Szalontai, Éva Szőke, Éva Sághy, Maja Payrits, Béla Kajtár, Krisztina Kovács, József László Környei, János Garai, András Garami, Anikó Perkecz, Levente Czeglédi and Zsuzsanna Helyes. Oestrogen-dependent up-regulation of TRPA1 and TRPV1 receptor proteins in the rat endometrium. J Mol Endocrinol December 7, 2015 JME-15-0184 doi: 10.1530/JME-15-0184

... After washing, sections were incubated in normal blocking solution, treated 160 with rabbit polyclonal anti-TRPV1 (Neuromics, Edina MN, USA)...

Souichi Kokubun, Tadasu Sato, Chikara Ogawa, Kai Kudo, Koju Goto, Yuki Fujii, Yoshinaka Shimizu, Hiroyuki Ichikawa. Distribution of TRPV1 and TRPV2 in the human stellate ganglion and spinal cord. Neuroscience Letters. Available online 29 January 2015. doi:10.1016/j.neulet.2015.01.074.

...The sections were incubated with rabbit antiserum against TRPV1 (1: 25,000, Neuromics, USA0...

Cai-hua Wu, Zheng-tao Lv, Yin Zhao, Yan Gao, Jia-qing Li, Fang Gao, Xian-fang Meng, Bo Tian, Jing Shi, Hui-lin Pan and Man Li. Electroacupuncture improves thermal and mechanical sensitivities in a rat model of postherpetic neuralgia. Molecular Pain 2013, 9:18 doi:10.1186/1744-8069-9-18

...Immunofluorescence labeling of TRPV1 receptors in left DRG (ips ilateral to EA treatment)and the spinal cord of vehicle-, RTX-, EA-, and sham EA-treated ra ts were performed to determine the effect of EA on RTX-induced deletion of TRPV1 recept ors. After 5 weeks of EA treatment (6 weeks after RTX injection), six rats in each group were deeply anesthetized with 10% chloralic hydras (3.5 ml/kg, i.p.) and transcardially perfuse d with 500 ml 37°C normal saline followed by 500 ml 4°C paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The lumbar L4-L6 segment of the spinal cord and DRG were quickly removed and postfixed for 2 hr in the same fixative solution and cryoprotected in 30% sucrose in 0.1 M phosphate buffer for 48 hr at 4°C. The sections were cut at 25 μm on a cryostat, which were mounted onto gelatin-coated slides and air dried overnight. The sections were rinsed in 0.1 M PBS and blocked for 1 hr with 5% donkey serum and 0.2% tween-20 in PBS, followed by incubation at 37°C for 1 hr then at 4°C overnight with the primary antibody (rabbit anti-TRPV1 N terminal, dilution 1:1000; Neuromics, Minneapolis, MN) diluted in PBS containing 5% donkey serum, 0.3% Triton X-100. Subsequently, sections were washed 4 tim es with 0.05% Tween-20 in PBS for 5 min, and incubated with a secondary antibody: donkey a nti-rabbit IgG conjugated with Dynight 594 (1:500; Jackson ImmunoResearch). Sections were washed 4 times with 0.05% Tween-20 in PBS for 5 min, and then coverslipped. Digital images were acquired using an Olympus BX51 fluorescence microscope (Olympus, Tokyo, Japan). Images were captured using a Qimaging Micropublisher RTV 5.0 microscope camera and QCapture Pro 6.0 software (Qimaging, TX, USA). A total of 5–6 sections from DRG and the spinal cord were randomly selected in each rat. The number of unmyeli nated axons in each view field was counted by an investigator in a blind fashion. The are a of TRPV1-positive central terminals in each view field was measured by using I mage J software (NIH, Bethesda,USA).

J.L. Saloman, M.-K. Chung, J.Y. Ro. P2X3 and TRPV1 functionally interact and mediate sensitization of trigeminal sensory neurons. Neuroscience, Volume 232, 1 March 2013, Pages 226-238.

...TRPV1 (1:1000; rabbit cat# RA10110; Neuromics, Edina, MN)...

Leanne M. Ramer, A. Peter van Stolk, Jessica A. Inskip, Matt S. Ramer and Andrei V. Krassioukov. Plasticity of TRPV1-expressing sensory neurons mediating autonomic dysreflexia following spinal cord injury. DOI=10.3389/fphys.2012.00257

For immunohistochemistry, slides were incubated in 10% normal donkey serum in PBS plus Triton X-100 (0.1%) for 20 min. We used five antibodies to delineate different subsets of nociceptors: these included antibodies raised against TRPV1 (Neuromics, Edina, MN, USA; 1:2,000)...Substance P (Neuromics, 1:1,000)...  β-III-tubulin (Neuromics; 1:500) – were used to label all neuronal profiles in the DRG.

Lee, Jongseok/ Saloman, Jami L. / Weiland, Gustave / Auh, Q-Schick / Chung, Man-Kyo / Ro, Jin Y. Functional interactions between NMDA receptors and TRPV1 in trigeminal sensory neurons mediate mechanical hyperalgesia in the rat masseter muscle. PAIN, 153 (7), p.1514-1524, Jul 2012 doi:10.1016/j.pain.2012.04.015

...sections were incubated overnight with primary antisera for TRPV1 (1:1000; rabbit polyclonal; Neuromics, Edina, MN), followed by 60 minutes with Daylight 488 goat antirabbit antiserum (1:250; Jackson Immunoresearch West)...

Jongseok Lee, Jami L. Saloman, Gustave Weiland, Q-Schick Auh, Man-Kyo Chung, Jin Y. Ro. Functional interactions between NMDA receptors and TRPV1 in trigeminal sensory neurons mediate mechanical hyperalgesia in the rat masseter muscle. doi.org/10.1016/j.pain.2012.04.015. PAIN Volume 153, Issue 7, July 2012, Pages 1514–1524.

...sections were incubated overnight with primary antisera for TRPV1 (1:1000; rabbit polyclonal; Neuromics, Edina, MN)...

Lintao Qua, Pu Zhang, Robert H. LaMotte and Chao Ma. Neuronal Fc-gamma receptor I mediated excitatory effects of IgG immune complex on rat dorsal root ganglion neurons. Brain, Behavior, and Immunity. doi:10.1016/j.bbi.2011.04.008.

...rabbit-anti-TRPV1, 1:1000, Neuromics...

T. Wu, L. Song, X. Shi, Z. Jiang, J. Santos-Sacchi and A.L. Nuttal. Effect of capsaicin on potassium conductance and electromotility of guinea pig outer hair cell. doi:10.1016/j.heares.2010.10.010

...anti-TRPV1 (rabbit polyclonal, RA10110, Neuromics, Edina, MN, USA) diluted to 1:500 with 1% BSA-PBS...antibody (TRPV-1) and its blocking peptide (104 M) (Neuromics, Edina, MN, USA)...

 Muthu D. Bhaskaran and Bret N. Smith. Effects of TRPV1 activation on synaptic excitation in the dentate gyrus of a mouse model of temporal lobe epilepsy. doi:10.1016/j.expneurol.2010.01.021

...with polyclonal VR1 N-terminus (1:2500) (Neuromics, Edina)...

Sonia K Bhangoo, Matthew S Ripsch, David J Buchanan, Richard J Miller and Fletcher A White. Increased chemokine signaling in a model of HIV1-associated peripheral neuropathy. Molecular Pain 2009, 5:48doi:10.1186/1744-8069-5-48.

...For some double-labeling experiments, slides were then incubated in anti-TRPV1 antibody (rabbit polyclonal, 1:1000; Neuromics), followed by incubation in secondary antibodies (donkey conjugated to CY5)...

Sacha A. Malin, Julie A. Christianson, Klaus Bielefeldt, and Brian M. Davis. TPRV1 Expression Defines Functionally Distinct Pelvic Colon Afferents. J. Neurosci., Jan 2009; 29: ; doi:10.1523/JNEUROSCI.3791-08.2009.

...Each section was stained with rabbit anti-TRPV1 (Neuromics)...

Sefika C Mizrak, Bart M Gadella, Hatice Erdost, Aytekin Ozer, Ana MM van Pelt, Federica MF van Dissel-Emiliani. Spermatogonial stem cell sensitivity to capsaicin: An in vitro study.  Reproductive Biology and Endocrinology 2008, 6:52 doi:10.1186/1477-7827-6-52. 743 - 752

Anti TRPV1 antibody staining: Bouin's fixed, paraffin embedded 5 um-thick rat testis sections were deparaffinized and boiled in a microwave oven (700 Watt) 3x10 min in sodium citrate buffer (0.1 mM,  pH=6) for antigen retrieval. All subsequent incubations were performed for 1 hour at  room temperature. The slides were then blocked with 5 % goat serum in 1 % BSA/PBS and incubated with the rabbit anti human - VR1 antibody (Neuromics, Edina, MN, USA; 1:500 in 1% BSA/PBS). Biotinilated goat anti-rabbit secondary antibody (BA-1000, Vector Labs; 1:200 in 1% BSA/PBS) was then applied. The ABC kit was finally used according to the manufacturer's instructions. Antibody reactivity was finally detected by diaminobenzidine staining (DAB, Sigma, St. Louis, MO, USA). Sections were counterstained with hematoxylin, dehydrated, mounted with Pertex and studied. Goat serum was applied on control sections.

SDS-PAGE and Western blotting: Protein lysates from the cell lines Gc-5spg and Gc-6spg and the control glioma cell line (A10-85) were prepared in RIPA buffer (PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS) including 1 mM phenylmethylsulfonylfluoride. Of each sample, 50 μg were separated on a 12% SDS-polyacrylamide gel and blotted onto a polyvinylidene fluoride membrane (Millipore Corp., Bedford, MA, USA). Western blots were blocked using Blotto-A, containing 5% Protifar (Nutricia, Zoetermeer, The Netherlands) in Tris-buffered saline (10 mM Tris; 150 mM NaCl, pH 7.6), including 0.05% Tween-20. Rabbit polyclonal anti-VR1 antibody (Neuromics) was diluted 1:1000 in Blotto-A and incubated for 1 h at room temperature. Blots were washed with Tris-buffered saline with 0.05% Tween-20. After incubation with goat anti-rabbit-HRP (P-0260 Dako Cytomation, 1:5000 inBlotto-A) secondary antibody for 1 h, blots were incubated with the electrochemiluminescence kit (ECL, Amersham Pharmacia Biotech, Little Chalfont, UK)and exposed to an x-ray film (RX-omat, Kodak, Chalone / Saone, France).

Alexander T. Stein, Carmen A. Ufret-Vincenty, Li Hua, Luis F. Santana, and Sharona E. Gordon. Phosphoinositide 3-Kinase Binds to TRPV1 and Mediates NGF-stimulated TRPV1 Trafficking to the Plasma Membrane. Published online Oct 30 2006. doi:10.1085/jgp.200609576 © The Rockefeller Press. 

Sharif Naeini R, Witty MF, Seguela P, Bourque CW (2006) An N-terminal variant of Trpv1 channel is required for osmosensory transduction. Nature Neuroscience. (1):93-8.

Aoki Y, Ohtori S, Takahashi K, Ino H, Douya H, Ozawa T, Saito T, and Moriya H (2005). Expression and Co-Expression of VR1, CGRP, and IB4-Binding Glycoprotein in Dorsal Root Ganglion Neurons in Rats: Differences Between the Disc Afferents and the Cutaneous Afferents. Spine 30(13):1496-1500.

Labrakakis C, Tong C, Weissman T, Torsney C and MacDermott A (2003). Localization and function of ATP and GABAA receptors expressed by nociceptors and other postnatal sensory neurons in rat. J Physiol 549(1):131-142.

Liapi A, and Wood JN (2005). Extensive co-localization and heteromultimer formation of the vanilloid receptor-like protein TRPV2 and the capsaicin receptor TRPV1 in the adult rat cerebral cortex.  Eur J Neurosci 22(4):825.

Nakatsuka T, Tsuzuki K, Ling JX, Sonobe H and Gu JG (2003). Distinct Roles of P2X Receptors in Modulating Glutamate Release at Different Primary Sensory Synapses in Rat Spinal Cord. J Neurophys 89: 3243-3252.

Rosenbaum T, Awaya M, and Gordon SE (2002). Subunit modification and association in VR1 ion channels. BMC Neurosci 2002, 3:4.

Zahner MR, Li DP, Chen SR, and Pan HL (2003). Cardiac vanilloid receptor 1-expressing afferent nerves and their role in the cardiogenic sympathetic reflex in rats. J Physiol 551(2):515-523. 

Zimov S and Yazulla S (2004). Localization of vanilloid receptor 1 (TRPV1/VR1)-like immunoreactivity in goldfish and zebrafish retinas: Restriction to photoreceptor synaptic ribbons. J Neurocytol 33:441–452.

Zwick M, Davis BM, Woodbury CJ, Burkett JH, Koerber HR, Simpson JF, and Albers KM (2002). Glial Cell Line-Derived Neurotrophic Factor is a Survival Factor for Isolectin B4-Positive, but not Vanilloid Receptor 1-Positive, Neurons in the Mouse. J Neurosci 22(10):4057-4065..

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VR1 (TRPV1)-Goat

Xiaoxue Peng, Keith Studholme, Martha P Kanjiya, Jennifer Luk, Diane Bogdan, Matthew W Elmes, Gregory Carbonetti, Simon Tong, , , Yu-Han Gary Teng, , , Robert C Rizzo, , , Huilin Li, , , Dale G Deutsch, , , Iwao Ojima, , , Mario J Rebecchi, Michelino Puopolo, Martin Kaczocha, Fatty-acid-binding protein inhibition produces analgesic effects through peripheral and central mechanisms. Molecular Pain. First published date: March-20-2017. 10.1177/1744806917697007

...Tissues were incubated overnight at 4C with primary antibodies diluted in PBS containing 0.3% Triton X-100 and 5% NDS. Primary antisera used were rabbit anti-FABP5 (BioVendor, #RD181060100, RRID:AB_344491) at 1:800 dilution in brain and 1:600 in DRGs, mouse anti-TRPV1 (UC Davis/NIH NeuroMab, #75-254, RRID:AB_11000725) at 1:400 dilution in brain tissue, goat anti-TRPV1 (Neuromics, #GT15129-100, RRID:AB_2209002) at 1:100 dilution in DRGs, mouse anti-peripherin (Millipore, #MAB5380, RRID:AB_2171352) at 1:75 dilution, goat anti-FAAH (LifeSpan Cat# LS-B4126-50 RRID:AB_10720351) at 1:50 in DRGs, and goat anti-CGRP (AbD Serotec Cat#1720-9007 RRID:AB_2290729) at 1:400 in DRGs. The sections were washed three times for 10 min each in PBS. The slides were incubated for 1 h at room temperature with secondary antibodies diluted in PBS with 0.3% Triton X-100 and 5% NDS....

Ki Seok Lee, Youping Zhang, Jamila Asgar, Q-Schick Auh, Man-Kyo Chung, Jin Y. Ro. Androgen receptor transcriptionally regulates μ-opioid receptor expression in rat trigeminal ganglia. Neuroscience. 2016.  doi:10.1016/j.neuroscience.2016.06.023

...For double-labeling immunohistochemistry, the sections were incubated overnight with primary antisera for AR, and incubated with Daylight 488 anti-rabbit antiserum (1:250, Jackson Immunoresearch) for 60 min at 37 °C for immunofluorescence. After staining for AR, the same sections were incubated overnight with primary antisera for either TRPV1 (1:1000, anti-goat, Neuromics, GT15129) or MOR (1:50, anti-goat, Santa Cruz, SC-27072) followed by 60 min with Cy-3 anti-goat antiserum (1:500; Jackson Immunoresearch) for immunofluorescence. We chose to examine AR in TRPV1- and MOR-positive cells since MOR is expressed primarily in peptidergic, TRPV1-positive neurons in sensory ganglia (Scherrer et al., 2009) and AR is required in MOR-positive neurons for transcriptional regulation of MOR expression. The primary antibody for TRPV1 or MOR was omitted from the processing of selected sections to control for non-specific background staining. Both TRPV1 and MOR primary antibodies have been characterized in previous studies (Czaja et al., 2008 and Wu et al., 2015)...

Ken Koda, Kana Hyakkoku, Koichi Ogawa, Keiko Takasu, Sunao Imai, Yusuke Sakurai, Masahide Fujita, Hiroko Ono, Miyuki Yamamoto, Isao Fukuda, Shoji Yamane, Atsushi Morita, Toshiyuki Asaki, Toshiyuki Kanemasa, Gaku Sakaguchi,& Yasuhide Morioka. Sensitization of TRPV1 by protein kinase C in rats with mono-iodoacetate-induced joint pain. Osteoarthritis and Cartilage. Published online: March 09, 2016. DOI: http://dx.doi.org/10.1016/j.joca.2016.02.010

(Edina, MN, USA), Goat, Polyclonal, 1:300, GT15129, Lot#401557. ... Isotype controls. Goat control IgG, Neuromics, Goat, Polyclonal, 1:300, GT15900, Lot#400923. ...

Deborah M. Hegarty, Sam M. Hermes, Tally M. Largent-Milnes, Sue A. Aicher. Capsaicin-responsive corneal afferents do not contain TRPV1 at their central terminals in trigeminal nucleus caudalis in rats. Journal of Chemical Neuroanatomy. Volumes 61–62, November 2014, Pages 1–12

...Goat anti-VR1 (TRPV1), N-terminus residues 4–21 of VR1 ( RASLDSEESESPPQENSC) 0.75 μg/ml, Catalog No. GT15129, Neuromics, Edina, MN, Alexa Fluor ® 647 donkey anti-goat, 1:800, Invitrogen/Life Technologies)...

Line Pourtau, Amarins Nieske Heeringa, Carole Rovere, Agnes Aubert, Jean-Louis Nahon, Sylvain Miraux, Jan Pieter Konsman. Increased circulating rather than spinal cytokines accompany chronic pain behaviors in experimental bone cancer and arthritis. Neuroimmunology and Neuroinflammation [serial online] 2014 [cited 2014 Nov 18];1:153-60]

...transient receptor potential vanilloid 1 (TRPV1; guinea pig antiserum diluted 1:500; Neuromics, Edina, MN, USA)...

Haijun Zhang, Jessica A. Boyette-Davis, Alyssa K. Kosturakis, Yan Li, Seo-Yeon Yoon, Edgar T. Walters, Patrick M. Dougherty. Induction of Monocyte Chemoattractant Protein-1 (MCP-1) and Its Receptor CCR2 in Primary Sensory Neurons Contributes to Paclitaxel-Induced Peripheral Neuropathy. The Journal of Pain Available online 31 May 2013. doi.org/10.1016/j.jpain.2013.03.012.

...transient receptor potential cation channel V1 (goat, 1:1000; Neuromics, Edina, MN...

Andrea M. Harrington, Patrick A. Hughes, Christopher M. Martin Jing Yang, Joel Castro, Nicole J. Isaacs, L. Ashley Blackshaw and Stuart M. Brierley. A novel role for TRPM8 in visceral afferent function. PAIN. doi:10.1016/j.pain.2011.01.027 

...goat-anti TRPV1 (1:200; GT15129 Neuromics Inc., Edina, MN)...

Hyun Yeong Kim, Kihwan Kim, Hai Ying Li, Gehoon Chunga, Chul-Kyu Park, Joong Soo Kim, Sung Jun Jung, Min Kyung Lee, Dong Kuk Ahn, Se Jin Hwang, Youngnam Kang, Alexander M. Binshtok, Bruce P. Bean, Clifford J. Woolf, Seog Bae Oh. Selectively targeting pain in the trigeminal system. Volume 150, Issue 1, Pages 29-40 (July 2010). 

...goat anti-TRPV1 (1:1000; Neuromics, USA)...

K. Czaja, G.A. Burns, and R.C. Ritter. Capsaicin-induced neuronal death and proliferation of the primary sensory neurons located in the nodose ganglia of adult rats. Neuroscience. 2008 June 23; 154(2): 621–630.

...VR1 Neuromics GT15129 Goat 1:200...

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(TRPV1) - mouse specific

Changming Wang, Zhongli Wang, Yan Yang, Chan Zhu, Guanyi Wu, Guang Yu, Tunyu Jian, Niuniu Yang, Hao Shi, Min Tang, Qian He, Lei Lan, Qin Liu, Yun Guan, Xinzhong Dong, Jinao Duan and Zongxiang Tang. Pirt contributes to uterine contraction-induced pain in mice. Molecular Pain 2015, 11:57 doi:10.1186/s12990-015-0054-x

... Dil-labeled sections were also incubated with rabbit anti-TRPV1 (1:1000; Neuromics, Edina, MN, USA) primary antibodies at 4 °C overnight. Then, the sections were incubated in Alexa Fluor-conjugated donkey anti-rabbit IgG (1:100, molecular probes, Eugene, OR, USA) secondary antibody at room temperature for 2 h...

Carl Weitlauf, Nicholas J. Ward, Wendi S. Lambert, Tatiana N. Sidorova, Karen W. Ho, Rebecca M. Sappington, and David J. Calkins. Short-Term Increases in Transient Receptor Potential Vanilloid-1 Mediate Stress-Induced Enhancement of Neuronal Excitation. The Journal of Neuroscience, 12 November 2014, 34(46): 15369-15381; doi: 10.1523/JNEUROSCI.3424-14.2014

...using a highly specific rabbit anti-TRPV1 (1:100; Neuromics)...

Marics I, Malapert P, Reynders A, Gaillard S, Moqrich A (2014) Acute Heat-Evoked Temperature Sensation Is Impaired but Not Abolished in Mice Lacking TRPV1 and TRPV3 Channels. PLoS ONE 9(6): e99828. doi:10.1371/journal.pone.0099828.

...TRPV1 antibody (1/1000 dilution, Neuromics)...

Dongyue Huang, Shuyang Li, Ajay Dhaka, Gina M Story and Yu-Qing Cao. Expression of the transient receptor potential channels TRPV1, TRPA1 and TRPM8 in mouse trigeminal primary afferent neurons innervating the dura. Molecular Pain 2012, 8:66 doi:10.1186/1744-8069-8-66.

...TRPV1 (Neuromics) were used at 1:1000...

Sarah E. Canetta, Edlira Luca, Elyse Pertot, Lorna W. Role,  David A. Talmage. Type III Nrg1 Back Signaling Enhances Functional TRPV1 along Sensory Axons Contributing to Basal and Inflammatory Thermal Pain Sensation. PLoS ONE 6(9): e25108. doi:10.1371/journal.pone.0025108.

...IHC: TRPV1 (guinea pig, 1:1000, GP14100 Neuromics) WB: TRPV1 (rabbit, 1:1000, RA14113 Neuromics)

Mahendra Bishnoi, Christine A Bosgraaf, Mruvil Abooj, Linlin Zhong, Louis S Premkumar. Streptozotocin-Induced Julie A. Christianson, Klaus Bielefeldt, Sacha A. Malin and Brian M. Davis. Neonatal colon insult alters growth factor expression and TRPA1 responses in adult mice. Pain Volume 151, Issue 2, November 2010, Pages 540-549.

...primary antiserum to TRPV1 (1:4000; Neuromics, Minneapolis, MN; cat# RA14113)...

Kenjiro Matsumoto, Emi Kurosawa, Hiroyuki Terui, Takuji Hosoya, Kimihito Tashima, Toshihiko Murayama, John V Priestley, and Syunji Horie Localization of TRPV1 and contractile effect of capsaicin in mouse large intestine: high abundance and sensitivity in rectum and distal colon. Am J Physiol Gastrointest Liver Physiol, Jun 2009; doi:10.1152/ajpgi.90578.2008

 ...TRPV1 antibody (mouse TRPV1 C-terminus; Neuromics Inc., Minneapolis, MN, USA) were 1:60...different anti-TRPV1 antibodies (1:60,000, Neuromics Inc., and rat TRPV1 C-terminus, 1:1,000...experiments, the antibody (1:60,000; Neuromics Inc.) was pre-incubated with 10 M of...

Rebecca M. Sappington, Tatiana Sidorova, Daniel J. Long, and David J. Calkins. TRPV1: Contribution to Retinal Ganglion Cell Apoptosis and Increased Intracellular Ca2+ with Exposure to Hydrostatic Pressure. Invest. Ophthalmol. Vis. Sci., Feb 2009; 50: 717 - 728.

...we used rabbit anti-mouse TRPV1 IgG (1:500; catalog number RA14113; Neuromics, Edina, MN) against the absolute C terminus of mouse TRPV1 (EDAEVFKDSMAPGEK)...

Ann M. Bode, Yong-Yeon Cho, Duo Zheng, Feng Zhu, Marna E. Ericson, Wei-Ya Ma, Ke Yao, and Zigang Dong. Transient Receptor Potential Type Vanilloid 1 Suppresses Skin Carcinogenesis.
Cancer Res., Feb 2009; 69: 905 - 913.

...dorsal skin samples (100 mum) were processed and immunostained. For the human skin cancer tissue array, we used anti-TRPV1 (Neuromics), anti-EGFR (Cell Signaling), and Alexa Fluor 488 and 647-conjugated secondary antibodies. Mouse skin samples were immunostained...

Rebecca M Sappington, Tatiana Sidorova, Daniel J Long, and David Calkins.  TRPV1: Contribution to Retinal Ganglion Cell Apoptosis and Increased Intracellular Ca2+ with Exposure to Hydrostatic Pressure. (Investigative Ophthalmology and Visual Science. )
© 2008 Investigative Ophthalmology and Visual Science  DOI:  10.1167/iovs.08-2321.

Katrin Schnizler, Leonid P. Shutov, Michael J. Van Kanegan, Michelle A. Merrill, Blake Nichols, G. Stanley McKnight, Stefan Strack, Johannes W. Hell, and Yuriy M. Usachev. Protein Kinase A Anchoring via AKAP150 Is Essential for TRPV1 Modulation by Forskolin and Prostaglandin E2 in Mouse Sensory Neurons. The Journal of Neuroscience, May 7, 2008, 28(19):4904-4917; doi:10.1523/JNEUROSCI.0233-08.2008.

Jens Hjerling-Leffler, Mona AlQatari,  Patrik Ernfors, and Martin Koltzenburg.  Emergence of Functional Sensory Subtypes as Defined by Transient Receptor Potential Channel Expression. The Journal of Neuroscience, March 7, 2007 • 27(10):2435–2443 • 2435.

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VR1 C-terminus (TRPV1)-Guinea Pig

Chen J, Varga A, Selvarajah S, Jenes A, Dienes B, Sousa-Valente J, Kulik A, Veress G, Brain SD, Baker D, Urban L, Mackie K, Nagy I. Spatial Distribution of the Cannabinoid Type 1 and Capsaicin Receptors May Contribute to the Complexity of Their CrosstalkSci Rep. 2016 Sep 22;6:33307. doi: 10.1038/srep33307. 

... Frozen samples were inserted into a double replica table and fractured into two pieces at −130 °C and replicated by carbon deposition (5 nm thick), platinum (2 nm), and carbon (15 nm) in a freeze-fracture replica machine (BAF 060; BAL-TEC, Lichtenstein). They were digested in a solution containing 2.5% SDS and 20% sucrose in 15 mM Tris-HCl at 80 °C for 18 h then at 60 °C for 18 h. The replicas were washed in 25 mM PBS containing 0.05% bovine serum albumin (BSA; Roth, Germany) and then incubated in a blocking solution containing 5% BSA for 1 h. Subsequently, replicas were incubated in a mixture of primary antibodies (TRPV1 raised in guinea pig against the 22 amino acids of the C terminus (YTGSLKPEDAEVFKDSMVPGEK; Neuromics, USA), the dilution was 1:1000 and CB1 receptor (Prof. K. Mackie, raised in goat; Veress et al. 2014), dilution was 1:4000) in 50 mM Tris-buffered saline (TBS) containing 1% BSA. After several washes, replicas were reacted with a mixture of gold-coupled donkey anti-guinea pig (for TRPV1; 12 nm) and rabbit anti-goat (for CB1, 5 nm) secondary antibodies (1:30; BioCell Research Laboratories, Cardiff, UK) in 25 mM TBS containing 5% BSA. The anti-TRPV1 antibody from Neuromics provided better staining in the replicas than then the anti-TRPV1 antibody from Chemicon, which we tried in a pilot study. They were then washed and picked up on 100-mesh grids. The specificity of the antibodies was tested on tissues dissected from TRPV1 and CB1 knock out mice


Sarah Krupp. Effects of neonatal capsaicin on formalin-induced ATF3 expression in the adult rat. University of Louisville. ThinkIR: The University of Louisville's Institutional Repository Electronic Theses and Dissertations. 8-2016

....Animal Perfusion and Tissue Processing:
Upon completion of all experiments, two days post Gformalin injections, animals were sacrificed with with an IP injection of 10 cc pentobarbital and transcardially perfused with paraformaldehyde (PFA) for tissue fixation as previously described (Xu)et al.,)2014). The L4 and L5 DRG were immediately dissected out and placed into 4% PFA for overnight fixation and transferred to 30% sucrose for a minimum of 4 days for cryoprotection. Tissue was then embedded using freezing medium and cut into 14μm sections for further analysis.

Immunohistochemistry:

L4GL5 DRG were stained for TRPV1, TRPA1, and ATF3 using the following antibodies: guinea pig anti-TRPV1 (1:1000,) Neuromics) GP14100) with donkey anti-guinea pig 405 (1:100 Jackson Immuno)706475148), rabbit anti-TRPA1(1:80,000,) Neuromics RA14135) with donkey anti-rabbit 488 (1:100) Jackson Immuno 711545152), and mouse anti-ATF3 (1:250) Abcam, ab58668) with donkey anti-mouse Cy3 (1:100) Jackson Immuno 715165151) preadsorbed  with 20 µl rat serum and 80 µl blocking serum to prevent non-specific binding.....

Obadah N. Austah, Nikita B. Ruparel, Michael A. Henry, Roberto J. Fajardo, James E. Schmitz, & Anibal Diogenes. Capsaicin-sensitive Innervation Modulates the Development of Apical Periodontitis. Journal of Endodontics. Aug 2016. DOI: http://dx.doi.org/10.1016/j.joen.2016.06.009

... Immunofluorescence staining was performed with primary antibodies against neurofilament heavy (NFH) (chicken at 1:1000; Abcam, Cambridge, UK) and TRPV1 (VR1; guinea pig at 1:1000; Neuromics Inc, Minneapolis, MN) followed by visualization of immunoreactivity with ...

Elaine D. Por, Jae-Hyek Choi & Brian J. Lund. Low-Level Blast Exposure Increases Transient Receptor Potential Vanilloid 1 (TRPV1) Expression in the Rat Cornea. Current Eye Research. Accepted: 13 Nov 2015. Published online: 06 Apr 2016. DOI:10.3109/02713683.2015.1122812

...Protein expression was determined on frozen, de-paraffinized sections using antibodies directed against TRPV1, CGRP (GP14100, RA24112;Neuromics, Edina, MN)...

Mruvil Abooj, Mahendra Bishnoi, Christine A. Bosgraaf and Louis S. Premkumar. Changes in Spinal Cord Following Inflammatory and Neuropathic Pain and the Effectiveness of Resiniferatoxin. The Open Pain Journal. 2016, 9, 1-14. DOI: 10.2174/1876386301609010001

...Immunofluorescence labeling of TRPV1 receptors and microglial marker (OX-42) and astrocyte marker (GFAP) in
spinal dorsal horn was performed on three vehicle- treated, three sham-operated, three CFA-treated and three CCI rats
to determine the effect of CFA-treatment and CCI on TRPV1 receptor expression and microglial activation in spinal
dorsal horns. Rats were deeply anesthetized with i.p. injection of ketamine (85 mg/kg) and xylazine (5 mg/kg) and
perfused intracardially with 100 ml ice-cold normal saline followed by 150 ml 4% paraformaldehyde in 0.01 M PBS
(pH 7.4). The lumbar segment of the spinal cord were quickly removed and post-fixed for 2 hr in the same fixative
solution and cryoprotected in 20% sucrose in 0.01 M PBS for 24 hr at 4°C. Following cryoprotection, 15 μm sections
were cut from lumbar portion of spinal cords using a microtome (Leica CM 1850, North Central Instruments Inc,
Plymouth MN, USA)

For TRPV1 receptor, microglial marker (OX-42) and glial fibrillary acidic protein (GFAP) immunostaining, thesections were rinsed in 0.1 M PBS and permeated with 0.1% triton X in 0.01 M PBS for 30 minutes. The sections werethen blocked in 10% normal donkey serum in PBS for another 30 minutes. The sections were then incubated overnightwith the primary antibody (TRPV1: Guinea anti-TRPV1 N terminal (dilution 1:1000) Neuromics, Minneapolis, MN,USA and microglial activation marker: Mouse anti-CD11b (OX- 42) (dilution 1:100), mouse anti-GFAP antibody(1:100), Novus Biologicals, Littleton, CO, USA) diluted in PBS containing 1% normal donkey serum, 0.1% Triton X-100. Subsequently, sections were rinsed in PBS and incubated (1 hour) with the secondary antibody (Rhodamine donkey anti guinea pig IgG (dilution 1:50), Jackson Immuno-research Laboratories, PA, USA) diluted in PBS containing 1% normal donkey serum, 0.1% Triton X-100. Sections were washed and fixed. A confocal microscope(Specifications: Intensity- 20%, Gain-1 (4 for microglia slides), Zoom-1, Offset-0, Scan speed 150-160 seconds, Filter and color tool-normal) was used to view the sections, and areas of interest were photo documented and quantified...

Erin M. Boisvert, Sandra J. Engle, Shawn E. Hallowell, Ping Liu, Zhao-Wen Wang & Xue-Jun Li. The Specification and Maturation of Nociceptive Neurons from Human Embryonic Stem Cells. Scientific Reports 5, Article number: 16821 (2015)doi:10.1038/srep16821

...Immunocytochemistry and quantification:
The coverslips were fixed using 4% paraformaldehyde (Sigma-Aldrich)16. Next, they were rinsed with PBS (Life Sciences), and were incubated with 0.2% Triton X-100 (Sigma-Aldrich) for 10 minutes and 10% normal donkey serum (Jackson ImmunoResearch Laboratories, Inc. West Grove, PA) for 1h before overnight incubation with primary antibodies in PBS containing 5% donkey serum at 4 °C. Antigen-antibody reactions were developed by the appropriate fluorescein-conjugated secondary antibodies (Invitrogen and Jackson ImmunoResearch Laboratories, Inc.). Nuclei were stained with Hoechst (Invitrogen). Primary antibodies used in this study included βIII-tubulin (Tuj1, 1:1000, mouse IgG, Developmental Studies Hybridoma Bank [DSHB]), TrpV1 (Neuromics, guinea pig IgG, 1:1000), Nav1.7 (Neuromics, mIgG, 1:750), P75 (Advanced Targeting Systems, mIgG, 1:200), P2X3 (Millipore, guinea pig IgG, 1:1000), TrkA (R&D, goat IgG, 1:100), and AP2α (DSHB, mIgG, 1:200). Images were collected using a Zeiss camera mounted onto Zeiss Axiovert 200M fluorescence microscope or Zeiss LSM 510 Meta confocal microscope (Carl Zeiss Inc., Thornwood, NY.). To confirm the specificity of the staining, negative controls without primary antibodies were included in which no positive fluorescence signals were observed....

Xue-Long Wang, Bin Tian, Ya Huang, Xiao-Yan Peng, Li-Hua Chen, Jun-Cheng Li & Tong Liu. Hydrogen sulfide-induced itch requires activation of Cav3.2 T-type calcium channel in mice. Scientific Reports 5, Article number: 16768 (2015) doi:10.1038/srep16768

...The tissue sections were blocked with 10% goat serum, and incubated over night at 4oC with the primary antibodies guinea pig anti-TRPV1 antibody (1:1000, Neuromics) (Edina, MN, USA)....

Pourtau L, Heeringa AN, Rovere C, Aubert A, Nahon JL, Miraux S, Konsman JP. Increased circulating rather than spinal cytokines accompany chronic pain behaviors in experimental bone cancer and arthritis. Neuroimmunology and Neuroinflammation 2014 [2014 Nov 18];1:153-60.]

...transient receptor potential vanilloid 1 (TRPV1; guinea pig antiserum diluted 1:500; Neuromics, Edina, MN, USA)...

S Kelly, R J Chapman, S Woodhams, D R Sagar, J Turner, J J Burston, C Bullock, K Paton, J Huang, A Wong, D F McWilliams, B N Okine, D A Barrett, G J Hathway, D A Walsh, V Chapman. Increased function of pronociceptive TRPV1 at the level of the joint in a rat model of osteoarthritis pain. Ann Rheum Dis doi:10.1136/annrheumdis-2013-203413.

...Rat spinal cord sections from MIA- and saline-treated rats (n=5/group) (see online supplemental methods) were incubated with a polyclonal guinea pig anti-TRPV1 antibody (1 : 500, Neuromics, Edina, Minnesota, USA catalogue number GP14100) and then with Alexa 568-conjugated goat anti-guinea pig secondary antibody (1:300, Molecular Probes). TRPV1 immunostaining was visualised with a Leica DMRB/DM4000 B fluorescence microscope and images were acquired using Openlab software (PerkinElmer)...

M. Shukla, R. Quirion, W. Ma. Reduced expression of pain mediators and pain sensitivity in amyloid precursor protein over-expressing CRND8 transgenic mice. doi.org/10.1016/j.neuroscience.2013.06.064.

...guinea pig anti-vallinoid receptor 1 (VR1) (C-terminal, 1:1000, Neuromics, Edina, MN, USA)...

M. Shukla, R. Quirion, W. Ma. Reduced expression of pain mediators and pain sensitivity in amyloid precursor protein over-expressing CRND8 transgenic mice. Neuroscience Volume 250, 10 October 2013, Pages 92–101.doi.org/10.1016/j.neuroscience.2013.06.064.

...guinea pig anti-vallinoid receptor 1 (VR1) (C-terminal, 1:1000, Neuromics, Edina, MN, USA)...

Dustin Green, Shivani Ruparel, Linda Roman, Michael A. Henry, Kenneth M. Hargreaves. Role of Endogenous TRPV1 Agonists in a Post-Burn Pain Model of Partial-Thickness Injury. PAIN, Available online 26 July 2013, ISSN 0304-3959,http://dx.doi.org/10.1016/j.pain.2013.07.040.

...This staining included the use of primary antibodies against TRPV1 (rat-specific guinea pig polyclonal from Neuromics, GP14100; 1:2000 dilution) in combination with primary antibodies against specific immune cell-type markers including...

Tong Liu, Temugin Berta, Zhen-Zhong Xu,Chul-Kyu Park,Ling Zhang, Ning Lü, Qin Liu, Yang Liu, Yong-Jing Gao, Yen-Chin Liu, Qiufu Ma, Xinzhong Dong, and Ru-Rong Ji. TLR3 deficiency impairs spinal cord synaptic transmission, central sensitization, and pruritus in mice. J Clin Invest. 2012 June 1; 122(6): 2195–2207. Published online 2012 May 8. doi:10.1172/JCI45414.

...guinea pig anti-TRPV1 antibody (1:1,000; Neuromics), guinea pig anti-SP antibody (1:1,000; Neuromics), rabbit anti-CGRP antibody (1 2,000; Neuromics)...


Iryna A. Khasabova,Sergey Khasabov, Justin Paz, Catherine Harding-Rose, Donald A. Simone, and Virginia S. Seybold. Cannabinoid Type-1 Receptor Reduces Pain and Neurotoxicity Produced by Chemotherapy. The Journal of Neuroscience, 16 May 2012, 32(20): 7091-7101; doi: 10.1523/​JNEUROSCI.0403-12.2012

...guinea pig anti-TRPV1 (1:500; VR1 C terminus; Neuromics)...

Robert P. Watson, Elliot Lilley, Moh Panesar, Gurdip Bhalay, Steven Langridge, Shin-Shay Tian, Conor McClenaghan, Anna Ropenga, Fanning Zeng, Mark S. Nash. Increased prokineticin 2 expression in gut inflammation: role in visceral pain and intestinal ion transport. Neurogastroenterology & Motility. Volume 24, Issue 1, pages 65–e12, January 2012.

...Formalin-fixed, wax-embedded tissues with no overt signs of pathology were purchased from Asterand (Detroit, MI, USA); informed consent had been sought and received from all donors. All tissues were used in accordance with the Human Tissue Act 2004 (UK)...guinea pig anti-TRPV1 antibody (GP14100 – Neuromics, Edina, MN, USA)...

Sarah E. Canetta, Edlira Luca, Elyse Pertot, Lorna W. Role, David A. Talmage. Type III Nrg1 Back Signaling Enhances Functional TRPV1 along Sensory Axons Contributing to Basal and Inflammatory Thermal Pain Sensation. PLoS ONE 6(9): e25108. doi:10.1371/journal.pone.0025108.

...IHC: TRPV1 (guinea pig, 1:1000, GP14100 Neuromics) WB: TRPV1 (rabbit, 1:1000, RA14113 Neuromics)

Mahendra Bishnoi, Christine A Bosgraaf, Mruvil Abooj, Linlin Zhong, Louis S Premkumar. Streptozotocin-Induced Early Thermal Hyperalgesia is independent of Glycemic State of Rats: Role of Transient Receptor Potential Vanilloid 1(TRPV1) and inflammatory mediators.   Molecular Pain 2011, 7:52 doi:10.1186/1744-8069-7-52. Published: 27 July 2011.

...The sections were then incubated overnight with the primary antibody (TRPV1: Guinea anti-TRPV1 (dilution 1:1000) Neuromics, Minneapolis, MN, USA)...

N. Schuelert, C. Zhang, A.J. Mogg, L.M. Broad, D.L. Hepburn, E.S. Nisenbaum, M.P. Johnson and J.J. McDougal. Paradoxical effects of the cannabinoid CB2 receptor agonist GW405833 on rat osteoarthritic knee joint pain. Osteoarthritis and Cartilage Volume 18, Issue 11, November 2010, Pages 1536-1543.

...primary antiserum to TRPV1 (1:4000; Neuromics, Minneapolis, MN; cat# RA14113)...

Mariusz Mucha, Lezanne Ooi, John E. Linley, Pawel Mordaka, Carine Dalle, Brian Robertson, Nikita Gamper, and Ian C. Wood Transcriptional Control of KCNQ Channel Genes and the Regulation of Neuronal Excitability.
J. Neurosci., Oct 2010; 30: 13235 - 13245 ; doi:10.1523/JNEUROSCI.1981-10.2010

...1:1000 guinea pig anti-TRPV1 (Neuromics)...

James P. Lund,Somayeh Sadeghi,Tuija Athanassiadis, Nadia Caram Salas, François Auclair, Benoît Thivierge, Isabel Arsenault, Pierre Rompré, Karl-Gunnar Westberg, and Arlette Kolta.Assessment of the Potential Role of Muscle Spindle Mechanoreceptor Afferents in Chronic Muscle Pain in the Rat Masseter Muscle. PLoS One. 2010; 5(6): e11131. Published online 2010 June 15. doi: 10.1371/journal.pone.0011131.

Reagents Referenced:

Guinea Pig TRPV1

TRPV1 Blocking Peptide

Guinea Pig 2X3

P2X3 Blocking Peptide

E. Matthew Hoffman, Ruben Schechter, and Kenneth E. Miller Fixative Composition Alters Distributions of Immunoreactivity for Glutaminase and Two Markers of Nociceptive Neurons, Nav1.8 and TRPV1, in the Rat Dorsal Root Ganglion. J. Histochem. Cytochem., Apr 2010; 58: 329 - 344. 

...For TRPV1 detection, polyclonal guinea pig antiserum (Neuromics Edina, MN) against a 22-amino-acid peptide corresponding to the C-terminal residues (YTGSLKPEDAEVFKDSMVPGEK) of rat TRPV1...

Hao Sun, De-Pei Li, Shao-Rui Chen, Walter N. Hittelman and Hui-Lin Pan. Sensing of Blood Pressure Increase by Transient Receptor Potential Vanilloid 1 Receptors on Baroreceptors. doi:10.1124/jpet.109.160473

...guinea pig anti-TRPV1, dilution 1:1000, Neuromics...

Noritaka Imamachi, Goon Ho Park, Hyosang Lee, David J. Anderson, Melvin I. Simon, Allan I. Basbaum, and Sang-Kyou Han TRPV1-expressing primary afferents generate behavioral responses to pruritogens via multiple mechanisms. PNAS, Jul 2009; 106: 11330 - 11335

...rabbit anti-PLC 3 (a gift from the Sternweis Lab at University of Texas Southwestern, Dallas) and 1:400 guinea pig anti-TRPV1 (Neuromics). Secondary antibodies were conjugated to Alexa Fluor 488 or 568 flurochromes (Molecular Probes). Using a Leica TCS-NT confocal...

Djalil Baiou , Peter Santha , Antonio Avelino , Ana Charrua , Timea Bacskai , Klara Matesz , Francisco Cruz , Istvan Nagy.  Neurochemical characterization of insulin receptor-expressing primary sensory neurons in wild-type and vanilloid type 1 transient receptor potential receptor knockout mice. J. Comp. Neurol. 2007, 503 (2), 334-347.

T. NAKAGAWA, K. WAKAMATSU, N. ZHANG,  S. MAEDA, M. MINAMI, M. SATOH AND S. KANEKO. INTRATHECAL ADMINISTRATION OF ATP PRODUCES LONG-LASTING ALLODYNIA IN RATS: DIFFERENTIAL MECHANISMS IN THE PHASE OF THE INDUCTION AND MAINTENANCE. Neuroscience 147 (2007) 445–455.

Amol M. Patwardhan, Nathaniel A. Jeske, Theodore J. Price, Nikita Gamper, Armen N. Akopian, and Kenneth M. Hargreaves. The cannabinoid WIN 55,212-2 inhibits transient receptor potential vanilloid 1 (TRPV1) and evokes peripheral antihyperalgesia via calcineurin. PNAS 103 (July 2006): 11393-11398.

Ahluwalia J, Rang H, and Nagy I (2002). The putative role of vanilloid receptor-like protein-1 in mediating high threshold noxious heat-sensitivity in rat cultured primary sensory neurons. Eur J Neurosci 16:1483-1489.

Hong S, and Wiley JW (2005). Early painful diabetic neuropathy is associated with differential changes in the expression and function of vanilloid receptor 1. J Biol Chem 280(1):618-27.

Hwang SJ, Burette A, and Valtschanoff JG (2003). VR1-positive primary afferents contact NK1-positive spinoparabrachial neurons. J Comp Neurol 460:255-265.

Liu M, Liu MC, Magoulas C, Priestly JV, Willmott NJ (2003). Versatile Regulation of Cytosolic Ca2+ by Vanilloid Receptor I in Rat Dorsal Root Ganglion Neurons. J Biol Chem 278(7): 5462-5472.

Price T, Louria M, Candelario-Soto D, Dussor G, Jeske N, Patwardhan A, Diogenes A, Trott A, Hargreaves K and Flores C (2005). Treatment of trigeminal ganglion neurons in vitro with NGF, GDNF or BDNF: effects on neuronal survival, neurochemical properties and TRPV1-mediated neuropeptide secretion. BMC Neurosci 6:4.

Wadachi R and Hargreaves KM (2006). Trigeminal Nociceptors Express TLR-4 and CD14: a Mechanism for Pain due to Infection. J Dent Res 85(1):49-53. 2003 Published by Elsevier Science B.V.

TRPV1-C and Skin

Phillip J. Albrecht, Scott Hines, Elon Eisenberg, Dorit Pud, Deborah R. Finlay, M. Kari Connolly, Michel Pare, Gudarz Davar, Frank L. Rice  Pathologic alterations of cutaneous innervation and vasculature in affected limbs from patients with complex regional pain syndrome. Pain, 2006 Feb;120(3):244-66. Epub 2006 Jan 19.  

...RA14113; Neuromics, Edina, MN)...


Sarah E. Canetta1, Edlira Luca, Elyse Pertot, Lorna W. Role1, David A. Talmage. Type III Nrg1 Back Signaling Enhances Functional TRPV1 along Sensory Axons Contributing to Basal and Inflammatory Thermal Pain Sensation. PLoS ONE 6(9): e25108. doi:10.1371/journal.pone.0025108.

...IHC: TRPV1 (guinea pig, 1:1000, GP14100 Neuromics) WB: TRPV1 (rabbit, 1:1000, RA14113 Neuromics)...

Hee Kyung Cho, M.D., Ph.D., So-Yeon Kim, Ph.D., Mi Jung Choi, Seung Ok Baek, M.D., Sang Gyu Kwak, Ph.D., and Sang Ho Ahn, M.D., Ph.D. The Effect of GCSB-5 a New Herbal Medicine on Changes in Pain Behavior and Neuroglial Activation in a Rat Model of Lumbar Disc Herniation. J Korean Neurosurg Soc. 2016 Mar; 59(2): 98-105. Published online 2016 Feb 29. doi: 10.3340/jkns.2016.59.2.98

...Samples were then treated with 10% normal donkey serum (NDS; Jackson Immunoresearch, Westgrove, PA, USA). Tissue sections were incubated overnight in a mixture of primary antibodies, that is, mouse anti-ionized calcium-binding adapter molecule 1 (Iba1) (Wako, Osaka, Japan; 1 : 1000), mouse anti-glial fibrillary acidic protein (GFAP) (BD Pharmingen, San Jose, CA, USA; 1 : 100), anti-transient receptor potential vanilloid type 1 (TRPV1) (Neuromics, Edina, MN, USA; 1 : 5000), and anti-calcitonin gene related peptide (CGRP) (Enzo, Farmingdale, NY, USA; 1 : 200). After reaction completion, tissues were rinsed with PBS (3×5 minutes), treated with 2% NDS for 15 minutes, and incubated with cy3-conjugated donkey anti-mouse (Jackson Im-munoresearch, PA, USA; 1 : 100), cy3-conjugated donkey anti-goat (Jackson Immunoresearch, PA, USA; 1 : 100), and Alexa 488-conjugated donkey anti-rabbit (Invitrogen, Eugene, OR, USA; 1 : 200) antibodies for 3 hours, rinsed with PBS, and mounted with Vectashield ...