T. Fuchigamia, O. Kakinohana, M.P. Hefferan, N. Lukacova, S. Marsala, O. Platoshyn, K. Sugahara, T.L. Yaksh, c, M. Marsal. Potent suppression of stretch reflex activity after systemic or spinal delivery of tizanidine in rats with spinal ischemia-induced chronic spastic paraplegia. doi:10.1016/j.neuroscience.2011.08.022.

...rabbit anti-α2A-m (immunogen sequence: KASRWRGRGNREKR; 1:100; Neuromics)...

Shao-Rui Chen,  Hao-Min Pan, Timothy E. Richardson, and Hui-Lin Pan. Potentiation of Spinal α2-Adrenoceptor Analgesia in Rats Deficient in TRPV1- Expressing Afferent Neurons. Published online 2007 March 24. doi: 10.1016/j.neuropharm.2007.03.009.

Double immunofluorescence labeling of TRPV1 and α2A- or α2C-ARs

To determine the co-localization and relationship between TRPV1-positive neurons and α2-ARs in both RTX- and vehicle-treated rats, doubling labeling of TRPV1 and α2A- or α2C-ARs in the spinal dorsal horn was performed on three vehicle- and three RTX-treated rats 4 wk after treatment. Under deep anesthesia with intraperitoneal sodium pentobarbital (60 mg/kg), each rat was intracardially perfused with 250 ml of 4% paraformaldehyde in 0.1 M PBS (pH 7.4) and 200 ml of 10% sucrose in 0.1 M PBS (pH 7.4). The lumbar spinal cord were quickly removed and postfixed in the same fixative solution for 2 hr at room temperature and cryoprotected in 30% sucrose in PBS for 48 hr at 4°C. The tissues were cut to 25 μm in thickness and collected free-floating in 0.1 M PBS. Sections intended for the labeling of TRPV1 and α2A-AR were incubated in a mixture of primary antibodies VR1 C-terminus (TRPV1, dilution: 1:1000; and rabbit anti-α2A-AR, dilution 1:100, Neuromics, Minneapolis, MN) for 2 hr at room temperature and overnight at 4°C. The sections used for the rabbit VR1 N-Terminus (TRPV1) double labeling of TRPV1 and α2C-AR were incubated in another mixture of primary antibodies (dilution 1:1000; and guinea pig anti-α2C-AR, dilution 1:500, Neuromics). The specificity of the α2A-AR and α2C-AR primary antibodies was demonstrated previously (Stone et al., 1998). Subsequently, sections labeled with TRPV1 and α2A-AR were incubated with a mixture of secondary antibodies (Biotin-SP conjugated to goat anti-rabbit IgG, dilution: 1:200, Jackson ImmunoResearch, West Grove, PA; and Alexa Fluor- 594 conjugated goat anti-guinea pig IgG, dilution 1:200, Molecular Probes, Eugene, OR). The remaining sections for TRPV1 and α2C-AR labeling were incubated in a different mixture of secondary antibodies (Biotin-SP conjugated to goat anti-guinea pig IgG, dilution: 1:200, Jackson ImmunoResearch; and Alexa Fluor-594 conjugated to goat anti-rabbit IgG, dilution: 1:200, Molecular Probes) for 2 hr at room temperature. Following the secondary antibody incubation, all sections were rinsed and incubated with streptavidin-conjugated horseradish peroxidase (PerkinElmer, Boston, MA) for 1 hr at room temperature. Next, the sections were incubated with FITC-tyramide (PerkinElmer, Boston, MA) for 10 min at room temperature, and then mounted on slides, dried, and coverslipped. The sections were examined on a laser scanning confocal microscope (Carl Zeiss, Jena, Germany), and areas of interest were photodocumented.


Li D, Atnip LM, Chen S, and Pan H. Regulation of Synaptic Inputs to Paraventricular-Spinal Output Neurons by 2 Adrenergic Receptors  J Neurophysiol 93: 393-402, 2005

... the labeling intensity of the first primary antibody (rabbit anti-alpha 2A polyclonal IgG antibody, Neuromics, Minneapolis, MN) was enhanced with ...

Pamela Rizk, Julio Salazar, Rita Raisman-Vozari, Marc Marien, Merle Ruberg, Francis Colpaert and Thomas Debeir. The Alpha2-Adrenoceptor Antagonist Dexefaroxan Enhances Hippocampal Neurogenesis by Increasing the Survival and Differentiation of New Granule  Cells. Neuropsychopharmacology 31, 1146-1157 (01 Jun 2006)

...guinea-pig anti-alpha2c-adrenoceptor (1:300; Neuromics, Bloomington, MN)...

...Confocal microscopy showed that noradrenergic afferents make contact with proliferating and differentiating cells, suggesting a direct noradrenergic influence on neurogenesis. Chronic systemic treatment of rats with dexefaroxan did not affect cell proliferation per se in the dentate gyrus (as monitored by bromodeoxyuridine-labeling), but promoted the long-term survival of newborn neurons by reducing apoptosis. Dexefaroxan treatment also enhanced the number and complexity of the dendritic arborizations of polysialated neural cell adhesion molecule-positive neurons. The trophic effects of dexefaroxan on newborn cells might involve an increase in brain-derived neurotrophic factor, which was upregulated in afferent noradrenergic fiber projection areas and in neurons in the granule cell layer. By promoting the survival of new endogenously formed neurons, dexefaroxan treatment represents a potential therapeutic strategy for maintaining adult neurogenesis in neurodegenerative conditions, such as Alzheimer's disease, that affect the hippocampus.