Optimization of non-isotopic 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 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.
Yao
R
R
Department of Ophthalmology, University of Medicine and Dentistry, New Jersey Medical School, Newark, New Jersey 07103, USA.
Sugino
I K
IK
Greulich
K M
KM
Ishida
M
M
Weier
H U
HU
Zarbin
M A
MA
D000818:Animals; D002415:Cats; D002467:Cell Nucleus / Q000648:ultrastructure; D004247:DNA / Q000302:isolation & purification; D015342:DNA Probes; D005557:Formaldehyde; D017403:In Situ Hybridization / Q000379:methods*; D008297:Male; D016612:Paraffin Embedding / Q000379:methods*; D010786:Photoreceptor Cells / Q000648:ultrastructure; D010857:Pigment Epithelium of Eye / Q000648:ultrastructure; D016133:Polymerase Chain Reaction; D012160:Retina / Q000648:ultrastructure*; D013194:Staining and Labeling; D014998:Y Chromosome / Q000648:ultrastructure*
D015342:DNA Probes; D005557:Formaldehyde; D004247:DNA