| Abstract No.: | C-D3128 |
| Country: | Canada |
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| Title: | δ OPIOID RECEPTOR IMMUNOHISTOCHEMISTRY AND WESTERN BLOT ANALYSIS IN MOUSE SPINAL CORDS: A COMPARATIVE STUDY BETWEEN ANTIBODIES FROM MULTIPLE SOURCES. |
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| Authors/Affiliations: | 1 Anne-Julie Chabot-Doré*; 2 Georges L. Wilcox; 1 Laura S. Stone;
1 McGill University, Montreal, QC, Canada; 2 University of Minnesota, Minneapolis, MN, USA
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| Content: | In the spinal cord, δ opioid (DOP) receptors are present in dorsal horn. Activation of DOP in the spinal cord results in analgesia. There is a lack of consensus around DOP receptor distribution in the spinal cord. Some groups have reported a pre-synaptic distribution in superficial lamina of the dorsal horn and a clear association of DOP receptors with substance P (SP) containing large dense core vesicles at the nerve terminal. In contrast, other groups have reported perikaria and dendritic labeling in the dorsal horn with a subcellular localization of DOP receptors on membrane-bound organelles such as the Golgi apparatus, the rough endoplasmic reticulum and clear vesicles. Discrepancies in the literature on DOP receptor localization depend on the antisera used, suggesting that variant DOP receptors have different antigenicity and are differentially distributed in neurons.
OBJECTIVE
The aim of this study is to a) compare several well characterized antibodies recognizing DOP that are known to generate distinct immunohistochemical labelling patterns despite targeting the same N-terminal epitope and b) to characterize a novel C-terminal antibody generated in chicken.
MATERIALS AND METHODS
Antibodies: Rb anti-DOPMN was originally produced and characterized by Dado et al. (1993) at the University of Minnesota (MN). Rb anti-DOPCHEM is a commercially available antisera from Chemicon (cat# AB1560, lot #LV1414387). Both rabbit antisera are directed against N-terminal residues 3-17 from the predicted DOP amino acid sequence (LVPSARAELQSSPLV). The chicken derived antisera (Ck anti-DOP) is directed against the C-terminal residues 346-360 (RPRQATTRERVTACT).
Western blot analysis will be performed on spinal cord membrane preparations from WT and DOP-KO mice. Immunofluorescence staining will be performed on WT and DOP-KO mice spinal cords with each DOP antisera and double staining will be performed with markers such as SP antisera.
RESULTS
Western blot analysis revealed the following bands: Rb anti-DOPMN: a 35 kDa doublet and bands at 45 kDa and 150 kDa; Rb anti-DOPCHEM: bands at 25 kDa and 35 kDa; Ck anti-DOP: a doublet at 35 kDa and a band at 90 kDa. Double immunofluorescence staining on WT mouse spinal cord showed an extensive co-localization with SP with the Rb anti-DOPMN but not the Rb anti-DOPCHEM.
CONCLUSION
To our knowledge, this is the first study that compares this set of well-characterized antisera raised against the same N-terminal epitope in mouse spinal cord by immunohistochemistry and western blot analysis. Our results in mice confirm results previously reported in rats. WB analysis of spinal cord extracts with the different antisera each show a distinct banding pattern, but a 35 kDa band is consistently labeled across the three antibodies tested. We report that immunohistochemistry labeling discrepancies are not explained by the amino acid sequence recognized by the antibody. We hypothesize that the different antisera may have variable affinity for different DOP receptor subtypes or variants.
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