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.
Images: Distinct distribution patterns of DORs in subsets of DRG neurons of mice. Immunostaining with antibodies against DOR13–17 [A: 1:30,000, antibody 1 (ab #1); DiaSorin and C: antibody 2 (ab #2); Neuromics] shows DORs in small DRG neurons and afferent fibers in spinal laminae I–II. This immunostaining pattern is abolished by the antiserum preabsorption or the deletion of Oprd1 exon 1. Reduction in immunostaining is quantitatively assayed by determining the percentage of positive DRG neurons (B; n = 6) and fluorescence intensity (Ifluo.) in the laminae I–II (D; n = 5). **P < 0.01; ***P < 0.001. (Scale bars: A and C, 40 μm.). DOR labeling (anti-DOR13–17, 1:30,000; DiaSorin) associated with vesicles in peptidergic small DRG neurons (E and F) is absent in Oprd1 exon 1-deleted mice (G). Colocalization of DORs and neuropeptides is shown by correlated peaks of Ifluo. measured along lines. (Scale bar: 8 μm.) (H) Immunostaining with antibodies against DOR12–18 (1:60,000; Alomone) shows the presence of DORs on the cell surface of large DRG neurons of mice. (Scale bar: 25 μm.) This staining pattern is abolished by preabsorption and is absent in Oprd1 exon 1-deleted mice. (Scale bar: 80 μm.) (I) Triple-immunostaining shows that DOR+ large DRG neurons contain neither SP nor CGRP. (Scale bar: 80 μm.)
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).
|Featured and Related Reagents:|
Mu Opioid Receptor-Rabbit
Mu Opioid Receptor-Guinea Pig
Delta Opioid Receptor 3-17
Delta Opioid Receptor 358-372
Delta Opioid Receptor 358-372
Kappa Opioid Receptor
phospho-Mu Opioid Receptor (Ser375)
All Opioid Receptor Antibodies
Pain and Inflammation
Neurotransmission Research Antibodies-GPCRs, Ligand Gated Ion
Channels, Biogenic Amines and more
i-Fect Transfection Kit -gene silencing
of DOR, NaV1.8 tetrodotoxin-resistant sodium channel, NTS2 and more
in-vitro and in vivo
Primary Neurons and Astrocytes-Primary
human, rat and mouse neurons and astrocytes