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P.S. Bernstein, P. Bhosale, M. Sharifzadeh, W. Gellermann, J.M. Frederick; Immunocytochemistry of Zeaxanthin–Binding Protein (GSTP1) in Monkey Retina . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2969.
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Uptake, metabolism, stabilization, and antioxidant functions of xanthophyll carotenoids in the vertebrate retina are mediated by specific xanthophyll binding proteins (XBPs). We recently identified and characterized glutathione S–transferase P1 (GSTP1) as a specific zeaxanthin–binding protein in the human retina (J. Biol. Chem. 2004; 279:49447). The distribution of GSTP1 in ocular tissues of monkey was investigated by immunocytochemistry and compared with resonance Raman imaging of macular pigment.
Confocal immunolocalization was performed on macular tissue from a juvenile Macaca fascicularis monkey whose eye was perfusion fixed with 2% paraformaldehyde. 12 µm–thick cryosections centered on the fovea were cut and blocked for 1 h using 10% normal donkey serum. Antibodies to GSTP1 (rabbit polyclonal) and cone arrestin (mouse monoclonal) were applied overnight at 4oC. After three ten–minute rinses, rhodamine– and FITC–conjugated secondary antibodies were applied for 2 h at room temperature. Sections were imaged using a Zeiss LSM 510 confocal microscope set to an optical slice of <0.9 µm. Resonance Raman imaging was performed on 20 µm–thick foveal sections to localize carotenoids with high resolution and chemical specificity.
Immunolocalization revealed that GSTP1 is present in neurons, but not glia, of the Macaca macula. Among photoreceptors, GSTP1 labeling was especially pronounced in cone myoid regions and, to a lesser extent, axons of Henle’s fiber layer and cone pedicles. GSTP1 immunoreactivity was also detected in perinuclear regions of horizontal, bipolar, and amacrine cells of the inner nuclear layer, and in ganglion cells. Colocalization of GSTP1 and CRALBP (a marker for Müller glial cells) was not observed. Quantitation of macular carotenoid pigments in this juvenile monkey's fellow eye by HPLC–APCI/MS indicated that the levels were 5–8 times lower than a typical adult human macula.
Observation of high levels of GSTP1 in primate macular plexiform layers correlated well with the distribution of macular carotenoids reported by Snodderly et al. and with our resonance Raman imaging studies. Interestingly, the myoid region of primate photoreceptors, which was labeled strongly by the anti–GSTP1 antibodies, corresponds anatomically to the carotenoid–rich oil droplets of the avian retina.
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