Detection of oligodendrocytes in tissue sections using PCR synthesis of digoxigenin-labeled probes.
Oligodendrocytes, the myelin-forming cells in the central nervous system, were visualized with excellent resolution at the light microscopic level using in situ hybridization (ISH). Digoxigenin (Dig)-tagged probes were synthesized and efficiently labeled by PCR. Specific probes to myelin genes were made by RT from brain total RNAs, followed by PCR with designed specific primers in the presence of Dig-11-dUTP. Probes specific to proteolipid protein (PLP), PLP and its isoform DM20 (PLP/DM20), and myelin oligodendrocyte glycoprotein (MOG) were synthesized and labeled. ISH was then applied on vibratomed tissue sections from mouse brains. Despite a low expression of MOG-specific and PLP-specific mRNAs in adult and newborn mouse brains, an oligodendrocyte population was detected. The specificity of Dig-labeled probes was confirmed with the double labeling of carbonic anhydrase II (CA II) and glial fibrillary acidic protein (GFAP) immunocytochemistry and ISH. This versatile and easy method for synthesis and labeling of specific probes to oligodendrocytes can be also applied to detect many other mRNAs in the nervous system and in other tissues.
Jalabi
Walid
W
UMR 7004 CNRS/ULP, Faculté de Médecine, Université Louis Pasteur, 11 rue Humann, 67085 Strasbourg, France.
Cerghet
Mirela
M
Skoff
Robert P
RP
Ghandour
M Said
MS
D000818:Animals; D001921:Brain / Q000166:cytology / Q000378:metabolism; D024402:Carbonic Anhydrase II / Q000378:metabolism; D015342:DNA Probes / Q000138:chemical synthesis* / Q000737:chemistry; D003856:Deoxyuracil Nucleotides / Q000737:chemistry; D004076:Digoxigenin / Q000031:analogs & derivatives* / Q000737:chemistry; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D051379:Mice; D009836:Oligodendroglia / Q000166:cytology* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012235:Rhodamines; D013116:Spinal Cord / Q000166:cytology / Q000378:metabolism
D015342:DNA Probes; D003856:Deoxyuracil Nucleotides; D005904:Glial Fibrillary Acidic Protein; D012235:Rhodamines; C070202:digoxigenin-11-deoxyuridine triphosphate; C009434:tetramethylrhodamine isothiocyanate; D024402:Carbonic Anhydrase II; D004076:Digoxigenin