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M. K. Wijono, W.-B. Im, G. Ruiz, E. WoldeMussie; Localization of 2 Receptors in Ocular Tissues. Invest. Ophthalmol. Vis. Sci. 2007;48(13):577.
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α2 adrenergic agonists are used for controlling IOP in the treatment of glaucoma. They have been shown to be neuroprotective to retinal cells in a variety of injury models. The aim of this study was to determine the distribution of the three α2 adrenergic receptors in ocular tissues using immunohistochemical techniques.
Antibodies were generated in albino rabbits against rat peptide residues from the C-terminal α2A, cytosolic loop of α2B, or the C-terminal α2C. Membrane proteins extracted from retinas or brains were used for western blot analysis. For immunostaining, eyes were fixed in 4% PFA with 10% picric acid, were processed, paraffin embedded and sectioned. Antigen retrieval was done on the tissue sections and they were immunostained with antibodies for α2A, α2B, and α2C.
Western blotting showed a specific band at the estimated molecular size for α2A, α2B and α2C. These bands were blocked by incubating ab with their respective peptide antigen. Immunostaining of α2A in rat retina was prominent in the plasma membrane of the ganglion and amacrine cells in the ganglion cell layer. Amacrine and horizontal cells in the inner nuclear layer also (INL) showed stronger staining than the rest of the inner nuclear cells. α2B staining was throughout the retina, from the fiber layer to the inner segment of the photoreceptors. The staining was mostly in the dendrites and axons. Intense staining was present in the innerplexiform layer (IPL), where there were 3 strata, proximal to the INL. α2C staining was present in the plasma membrane of the photoreceptor cells and the inner segment. All α2A, 2B, and 2C staining were blocked when the ab was preincubated with the respective peptide antigen.
Both immunoblotting and immunohistochemistry showed the presence of all 3 adrenergic receptor subtypes in the retina of the rat.
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