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UMB-3, a novel rabbit monoclonal antibody, for assessing μ-opioid receptor expression in mouse, rat and human
Amelie Lupp1, Nadine Richter, Christian Doll
1Department of Pharmacology and Toxicology, University Hospital, Friedrich Schiller University, D-07747 Jena, Germany.
Background:
The immunohistochemical localization of the μ-opioid receptor (MOR, MOP) has been studied in detail in mouse and rat brain using a variety of polyclonal antibodies. However, biochemical analysis of the MOR signaling complex in vivo has been hampered by the lack of suitable monoclonal antibodies for efficient immunoprecipitation of the receptor protein from native sources. Moreover, previous immunohistochemical investigations were restricted to frozen sections from perfusion-fixed rodent brain, largely due to the limited availability of MOR antibodies that effectively stain paraffin-embedded tissues.
Methods:
Here, we extensively characterized the novel rabbit monoclonal anti-MOR antibody UMB-3 using transfected cells and MOR-deficient mice. UMB-3 was also subjected to a comparative immunohistochemical study of formalin-fixed, paraffin-embedded mouse and rat organ samples as well as human normal and neoplastic tissues.
Results:
Specificity of UMB-3 was demonstrated by detection of a broad band migrating at M(r) 70,000-80,000 in immunoprecipitates from crude brain homogenates of MOR+/+ mice but not of MOR⁻/⁻ mice; cell surface staining of MOR-transfected cells; translocation of MOR receptor immunostaining after agonist exposure; distinct immunostaining of neuronal cell bodies and fibers in MOR-expressing brain regions; absence of staining in MOR-deficient mice; and abolition of tissue immunostaining by preadsorption of UMB-3 with its immunizing peptide.
Conclusions:
The rabbit monoclonal antibody UMB-3 is an excellent tool for immunoprecipitation of MOR from native sources as well as for immunohistochemical staining of MOR in paraffin-embedded tissue samples of rodent and human origin.
Insights
A new monoclonal antibody, UMB-3, effectively detects the μ-opioid receptor (MOR) in various tissues. This antibody enables both immunoprecipitation and immunohistochemical staining of MOR in paraffin-embedded samples from rodents and humans.
Area of Science:
- Neuroscience
- Molecular Biology
- Immunology
Background:
- Immunohistochemical studies of the μ-opioid receptor (MOR) in rodent brains have limitations.
- Lack of suitable monoclonal antibodies hinders in vivo biochemical analysis of the MOR signaling complex.
- Previous methods were restricted to frozen sections due to poor staining of paraffin-embedded tissues.
Purpose of the Study:
- To characterize a novel rabbit monoclonal anti-MOR antibody, UMB-3.
- To evaluate UMB-3 for immunoprecipitation and immunohistochemical staining.
- To assess UMB-3's utility in paraffin-embedded tissues from various species.
Main Methods:
- Extensive characterization of UMB-3 using transfected cells and MOR-deficient mice.
- Comparative immunohistochemical study of formalin-fixed, paraffin-embedded rodent and human tissues.
- Specificity validation through immunoprecipitation and peptide preadsorption.
Main Results:
- UMB-3 specifically detected MOR in immunoprecipitates from wild-type mouse brain homogenates.
- Antibody showed cell surface staining, agonist-induced translocation, and distinct neuronal staining patterns.
- Staining was absent in MOR-deficient mice and abolished by peptide preadsorption.
Conclusions:
- The rabbit monoclonal antibody UMB-3 is a valuable tool for MOR research.
- UMB-3 facilitates immunoprecipitation of MOR from native sources.
- UMB-3 enables reliable immunohistochemical staining of MOR in paraffin-embedded rodent and human tissues.
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