UMB-3, a novel rabbit monoclonal antibody, for assessing μ-opioid receptor expression in mouse, rat and human formalin-fixed and paraffin-embedded tissues.
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.
Lupp
Amelie
A
Department of Pharmacology and Toxicology, University Hospital, Friedrich Schiller University, D-07747 Jena, Germany.
Richter
Nadine
N
Doll
Christian
C
Nagel
Falko
F
Schulz
Stefan
S
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism*; D000915:Antibody Affinity; D001921:Brain / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D002478:Cells, Cultured; D005260:Female; D005404:Fixatives / Q000737:chemistry; D005557:Formaldehyde / Q000737:chemistry; D015870:Gene Expression; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D047468:Immunoprecipitation / Q000379:methods; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D009369:Neoplasms / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D016612:Paraffin Embedding; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D017450:Receptors, Opioid, mu / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D016707:Tissue Fixation; D014162:Transfection
D000911:Antibodies, Monoclonal; D005404:Fixatives; D017450:Receptors, Opioid, mu; D005557:Formaldehyde