OPTIMIZATION OF NONISOTOPIC IN-SITU HYBRIDIZATION - DETECTION OF THE Y-CHROMOSOME IN PARAFORMALDEHYDE-FIXED, WAX-EMBEDDED CAT RETINA

A technique was developed to detect the Y chromosome in paraformaldehy de-fixed diethylglycoldiesterate-embedded cat retina. The Y chromosome specific DNA probe was labeled with digoxigenin through polymerase ch ain reaction incorporation. After treatment of paraformaldehyde-fixed, diethylglycoldiesterate-embedded tissue sections with deoxyribonuclei c acid decondensation and proteolytic digestion, non-fluorescent, non- isotopic in situ hybridization was performed on the retina sections. M ost extensive treatment was required for the outer nuclear layer while the inner nuclear layer required more extensive treatment than the re tinal pigment epithelial cells. Under optimal pretreatment conditions, the male cat retina displayed black spots which specifically localize d at the periphery of the nuclei, while the female cat retina showed n egative staining for the Y chromosome specific probe. The technique al lows observation of the Y chromosome signal with preservation of retin al morphology and thus may be a valuable tool to discriminate donor ce lls in retinal pigment epithelial cell and photoreceptor cell transpla nts. (C) 1998 Academic Press Limited.