Opioid Receptors-Mu, Kappa and Delta

2010

Ying He, Cheng Yang, Chelsea M. Kirkmire, and Zaijie Jim Wang. Regulation of Opioid Tolerance by let-7 Family MicroRNA Targeting the µ Opioid Receptor. The Journal of Neuroscience, July 28, 2010, 30(30):10251-10258; doi:10.1523/JNEUROSCI.2419-10.2010

Western blot analysis. Western blot analysis was performed as previously described (Tang et al., 2006) using the anti-µ opioid receptor antibody (1:1000; Neuromics). The expression of β-actin was similarly determined from the same blots using a monoclonal antibody (1:10,000; Sigma).

For immunofluorescence analysis, the antibody for hDcp1a (Santa Cruz Biotechnology) and MOR were used at 1:500 and 1:5000 dilutions, respectively. Secondary anti-goat and anti-mouse antibodies labeled with Alexa 488 and Alexa 594 fluorochromes (Invitrogen), respectively, were used at 1:500 dilutions.

Hai-Bo Wanga, Bo Zhaoa, Yan-Qing Zhonga, Kai-Cheng Li, Zi-Yan Li, Qiong Wang, Yin-Jing Lua, Zhen-Ning Zhang, Shao-Qiu He, Han-Cheng Zheng, Sheng-Xi Wu, Tomas G. M. Hökfelt, Lan Baob, and Xu Zhanga. Coexpression of δ- and μ-opioid receptors in nociceptive sensory neurons. PNAS July 20, 2010 vol. 107 no. 29 13117-13122.

 Immunostaining. Adult rats, mice, and Oprd1 exon 1-deleted mice were fixed. Cryostat sections of L4 and L5 DRGs and spinal cord segments were processed for immunofluorescence staining (13) with Rb anti-DOR13–17 (1:2,000–1:60,000; DiaSorin and 1:4,000–1:60,000; Neuromics), Rb anti-DOR12–18 (1:30,000–1:120,000; Alomone), Rb anti-DOR1358–372 (1:1,000–1:2,000; Lifespan Biosciences), Rb anti-MOR (1:1,000; Neuromics); guinea pig anti-SP (1:500; Neuromics), and mouse anti-CGRP (1:1,000; Biogenesis) antibodies. IB4-labeling was carried out with fluorescein-labeled GSL I-IB4 (1:200). The Myc-DOR1–transfected HEK293 cells and neurons were fixed and processed with mouse anti-Myc antibodies (1:500; DSHB). Nuclear DAPI staining was used to indicate HEK293 cells in control experiments.

 Immunoblotting.The samples were processed for SDS/PAGE, transferred, probed with Rb antibodies against MOR (1:500; Neuromics), phospho-DOR1 (1:1,000; Neuromics), phospho-MOR (1:1,000; Neuromics), Myc (1:500; DSHB), Flag (1:1,000; Sigma), or actin (1:50,000; Chemicon) and visualized with enhanced chemiluminescence (19).

2009

Liping Xu1, Josef Vagner, Jatinder Josan, Ronald M. Lynch, David L. Morse, Brenda Baggett, Haiyong Han, Eugene A. Mash, Victor J. Hruby and Robert J. Gillies. Enhanced targeting with heterobivalent ligands. Published OnlineFirst August 2009. doi: 10.1158/1535-7163.MCT-08-1183. Mol Cancer Ther August 2009 8; 2356.

 .... Antibodies for δOR (mouse; Neuromics; RA10101)...

Dominika Labuz, Yvonne Schmidt, Anja Schreiter, Heike L. Rittner, Shaaban A. Mousa and Halina Machelska. Immune cell–derived opioids protect against neuropathic pain in mice. J. Clin. Invest. doi:10.1172/JCI36246. Copyright © 2009, The American Society for Clinical Investigation

κ-opioid receptor (1:800, Neuromics)

2008

Phoebe Dewing, Amy Christensen, Galyna Bondar and Paul Micevych. PKC signaling in the hypothalamic arcuate nucleus regulates sexual receptivity in female rats. Endocrinology. First published ahead of print July 24, 2008 as doi:10.1210/en.2008-0847.

...For MOR internalization, rabbit primary antibodies directed against MOR (1:24,000; Neuromics, Minneapolis, MN) were used. Sections processed for fluorescence were incubated in blocking buffer (Tyramide Signal Amplification kit, NEN Life Science Products, Boston, MA), and then in biotin conjugated goat anti-rabbit IgG (Vector Laboratories)...

Marta Pinto, Marta Sousa, Deolinda Lima, Isaura Tavares. Participation of -opioid, GABAB, and NK1 receptors of major pain control medullary areas in pathways targeting the rat spinal cord: Implications for descending modulation of nociceptive transmission. Volume510 Issue 2 Pages 175 - 187 Published Online: 9 Jul 2008. Copyright © 2008 Wiley-Liss, Inc., A Wiley Company.

...The antibody against MOR (Neuromics; Cat No. RA10104, Lot No 400058)...

Sasaki, Atsushi a; Nakashima, Yasutaka a; Takasaki, Ichiro c; Andoh, Tsugunobu a; Shiraki, Kimiyasu b; Kuraishi, Yasushi a d. Morphine inhibits herpetic allodynia through [mu]-opioid receptors induced in A[beta]-fiber neurons. Neuroreport. 19(9):975-979, June 11, 2008

...The dorsal root ganglion (DRG) at the L5 level was removed; transcardial perfusion, postfixation, and cryoprotection were done as described previously [13]. They were cut on a freezing microtome (Leica CM 3050S IV, Nussloch, Germany) at a 16-mm thickness. After being treated with a blocking solution (1.5% fetal bovine serum and 0.2% Triton X-100 in the phosphate-buffered saline) for 30 min at room temperature, sections were incubated with rabbit anti-MOR antibody (1 : 500; Neuromics Inc., Minneapolis, Minnesota, USA) at 41oC for 2 days. After washing, the sections were reacted with Alexa 488-conjugated donkey anti-rabbit immunoglobulin G antibody (1 : 1000; Molecular Probes Inc., Eugene, Oregon, USA) for 2 h at room temperature.Fluorescence signals were observed using Radiance 2000 confocal system (Bio-Rad Microscopy Division, Cambridge, Massachusetts, USA)...

Zhang, Guohua M.D., Ph.D.; Mohammad, Husam; Peper, Brad D.; Raja, Srinivasa M.D; Wilson, Steven P. Ph.D; Sweitzer, Sarah M. Ph.D.  Enhanced Peripheral Analgesia Using Virally Mediated Gene Transfer of the [mu]-Opioid Receptor in Mice.
Anesthesiology - Volume 108, Issue 2 (February 2008)  -  Copyright © 2008 American Society of Anesthesiologists, Inc.

...Blocking was followed by a 24-h incubation at 4°C in polyclonal rabbit anti-mu-OR (1:1,000; Neuromics, Minneapolis, MN)...

2006-2007

Sarah L. Poole, Jim Deuchars, David I. Lewis and Susan A. Deuchars. Subdivision-Specific Responses of Neurons in the Nucleus of the Tractus Solitarius to Activation of Mu-Opioid Receptors in the Rat. J Neurophysiol 98: 3060-3071, 2007. First published September 26, 2007; doi:10.1152/jn.00755.2007 0022-3077/07

...Sections required for electron microscopy were permeabilized in 50% ethanol for 30 min at room temperature, washed in PBS then incubated in a H2O2 solution for 30 min to block endogenous peroxidase activity. Sections for light microscopy were not treated with 50% ethanol for permeabilization, but had 0.1% TritonX100 added to the primary antibody solution. Tissue processing for light and electron microscope viewing has been described in detail previously (Deuchars et al. 2001a). Briefly, sections were incubated in polyclonal antiserum corresponding to residues 1359–1403 of the carboxy-terminus of the rat mu-opioid receptor (Neuromics, Minneapolis, USA), 1:2000 in PBS. This was localized with secondary biotinylated donkey anti-rabbit (1:200), prior to detection with extra-avidin peroxidase (1:1500) and visualization with DAB. Sections required for light microscopy were dehydrated in increasing concentrations of ethanol and mounted in DPX. Sections for electron microscopy were postfixed in 0.5% osmium tetra oxide and dehydrated in graded ethanol concentrations, 50%, 70%, 90% and 100%. Sections were immersed in Durcupan ACM resin for 12–18 h, mounted on slides, cover-slipped then placed in the oven at 60°C for 48 h to polymerize the resin. Slices were viewed at the light and electron microscope level...

Phoebe Dewing, Marissa I. Boulware,Kevin Sinchak, Amy Christensen, Paul G. Mermelstein,  and Paul Micevych. Membrane Estrogen Receptor- Interactions with Metabotropic Glutamate Receptor 1a Modulate Female Sexual Receptivity in Rats. doi:10.1523/JNEUROSCI.0592-07.2007.

....... rabbit primary antibodies directed against MOR (1:24000; Neuromics, Minneapolis) ...

S.-R. Chen, A. Prunean, H.-M. Pan, K.L. Welker and H.-L. Pan. Resistance to morphine analgesic tolerance in rats with deleted transient receptor potential vanilloid type 1-expressing sensory neurons. doi:10.1016/j.neuroscience.2006.12.016

.... antibodies (rabbit anti-μ opioid receptors, dilution 1:1000, Neuromics; and guinea-pig anti-TRPV1, ...

Eunhae Kim, Amy L. Clark , Alexi Kiss, Jason W. Hahn, Robin Wesselschmidt. MU AND KAPPA OPIOIDS INDUCE THE DIFFERENTIATION OF EMBRYONIC STEM CELLS TO NEURAL PROGENITORS JBC Papers in Press. Published on September 1, 2006 as Manuscript M603862200

2003-2005

Thomas F. Finnegan, Shao-Rui Chen, and Hui-Lin Pan. Effect of the µ Opioid on Excitatory and Inhibitory Synaptic Inputs to Periaqueductal Gray-Projecting Neurons in the Amygdala. JPET 312:441-448, 2005.

...The second primary antibody, rabbit anti-µ receptor (1:2000; Neuromics, Northfield, MN), was then applied for 2 h at room temperature and 24 h at 4°C. Sections were rinsed in Tris-buffered saline and 0.05% Tween 20 and exposed to the horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:100; Jackson ImmunoResearch Laboratories Inc.) for 90 min at room temperature...

Byron C. Yoburn*, Vishal Purohit, Kaushal Patel, Qiuyu Zhang. Opioid agonist and antagonist treatment differentially regulates immunoreactive Mu-opioid receptors and dynamin-2 in vivo. European Journal of Pharmacology 498 (2004) 87–96

...Primary Antibody (Mu-opioid receptor, 1:500; Neuromics, Northfield, ... Western blot protocol...

Tara A. Macey, Janet D. Lowe, and Charles Chavkin. Mu Opioid Receptor Activation of ERK1/2 Is GRK3 and Arrestin Dependent in Striatal Neurons. Published online 2006 September 18. doi: 10.1074/jbc.M60427820. 

..guinea pig anti-mu opioid receptor (1/1000 dilution, Neuromics, Minneapolis, MN)... 

Shuxing Wang, Grewo Lim, Liling Yang, Qing Zeng, Backil Sung, J.A. Jeevendra Martyn and Jianren Mao.

A rat model of unilateral hindpaw burn injury: Slowly developing rightwards shift of the morphine dose–response curve. doi:10.1016/j.pain.2005..03.044.

...MOR (1:2000; Rabbit anti-rat MOR, Neuromics Inc.)...

Wu Z, Chen S and Pan H. Differential Sensitivity of N- and P/Q-Type Ca2+ Channel Currents to a µ Opioid in Isolectin B -Positive and -Negative Dorsal Root Ganglion Neurons. JPET 311:939-947, 2004 

...Sections were incubated with the primary antibody (rabbit anti-µ opioid receptor, dilution 1:1000; Neuromics... 

Truong W, Cheng C, Xu Q, Li X, and Zochodne, D. Mu Opioid receptors and analgesia at the site of a peripheral nerve injury Annals of Neurology Volume 53, Issue 3 , Pages 366 - 375

Chen S and Pan H. Antinociceptive Effect of Morphine, but not Mu Opioid Receptor Number, Is Attenuated in the Spinal Cord of Diabetic Rats. Anesthesiology 2003; 99:1409–14. 

...sections were then incubated with the primary antibody (rabbit antibody to mu opioid receptor; dilution, 1:1,000; Neuromics... 

Jones T, Sweitzer S, Wilson S, and Yeomans D. (2003). Afferent fiber-selective shift in opiate potency following targeted opioid receptor knockdown. Pain 106 (2003) 365–371 

...antisera solution (1:1000) against the mOR (Neuromics...