Yao R, Sugino I K, Greulich K M, Ishida M, Weier H U, Zarbin M A
Department of Ophthalmology, University of Medicine and Dentistry, New Jersey Medical School, Newark, New Jersey 07103, USA.
Exp Eye Res. 1998 Feb;66(2):223-30. doi: 10.1006/exer.1997.0427.
A technique was developed to detect the Y chromosome in paraformaldehyde-fixed diethylglycoldiesterate-embedded cat retina. The Y chromosome specific DNA probe was labeled with digoxigenin through polymerase chain reaction incorporation. After treatment of paraformaldehyde-fixed, diethylglycoldiesterate-embedded tissue sections with deoxyribonucleic acid decondensation and proteolytic digestion, non-fluorescent, non-isotopic in situ hybridization was performed on the retina sections. Most extensive treatment was required for the outer nuclear layer while the inner nuclear layer required more extensive treatment than the retinal pigment epithelial cells. Under optimal pretreatment conditions, the male cat retina displayed black spots which specifically localized at the periphery of the nuclei, while the female cat retina showed negative staining for the Y chromosome specific probe. The technique allows observation of the Y chromosome signal with preservation of retinal morphology and thus may be a valuable tool to discriminate donor cells in retinal pigment epithelial cell and photoreceptor cell transplants.
开发了一种在经多聚甲醛固定、二乙二醇二乙酯包埋的猫视网膜中检测Y染色体的技术。Y染色体特异性DNA探针通过聚合酶链反应掺入用洋地黄毒苷标记。在用脱氧核糖核酸解聚和蛋白水解消化处理多聚甲醛固定、二乙二醇二乙酯包埋的组织切片后，对视网膜切片进行非荧光、非同位素原位杂交。外层神经核层需要最广泛的处理，而内核层比视网膜色素上皮细胞需要更广泛的处理。在最佳预处理条件下，雄性猫视网膜显示出黑点，这些黑点特异性地定位在细胞核周边，而雌性猫视网膜对Y染色体特异性探针呈阴性染色。该技术能够在保留视网膜形态的情况下观察Y染色体信号，因此可能是区分视网膜色素上皮细胞和光感受器细胞移植中供体细胞的有价值工具。
