In situ hybridization: use of 35S-labeled probes on paraffin tissue sections.
The following protocol is for radioactive in situ hybridization detection of RNA using paraffin-embedded tissue sections on glass microscope slides. Steps taken to inhibit RNase activity such as diethyl pyrocarbonate (DEPC) treatment of solutions and baked glassware are unnecessary. The tissue is fixed using 4% paraformaldehyde, hybridized with (35)S-labeled RNA probes, and exposed to nuclear-track emulsion. The entire procedure takes 2-3 days prior to autoradiography. The time required for autoradiography is variable with an average time of 10 days. Parameters that affect the length of the autoradiography include: (1) number of copies of mRNA in the tissue, (2) incorporation of label into the probe, and (3) amount of background signal. Additional steps involved in the autoradiography process, including development of the emulsion, cleaning of the microscope slides, counterstaining of the tissue, and mounting coverslips on the microscope slides, are discussed. In addition, a general guide to the interpretation of the in situ results is provided.
Micales
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BK
Anatomy Department, University of Wisconsin Medical School, 1300 University Avenue, Madison, Wisconsin 53706, USA.
Lyons
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GE
D000089:Acetic Anhydrides / Q000494:pharmacology; D000818:Animals; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004622:Embryo, Mammalian / Q000378:metabolism / Q000473:pathology; D004625:Embryo, Nonmammalian; D019286:Endopeptidase K / Q000378:metabolism; D004983:Ethanolamines / Q000494:pharmacology; D017403:In Situ Hybridization / Q000379:methods*; D015335:Molecular Probes; D009693:Nucleic Acid Hybridization; D010232:Paraffin / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D013462:Sulfur Radioisotopes / Q000494:pharmacology*; D013997:Time Factors
D000089:Acetic Anhydrides; D004983:Ethanolamines; D015335:Molecular Probes; D012333:RNA, Messenger; D013462:Sulfur Radioisotopes; C031800:acetic anhydride; D010232:Paraffin; C009546:triethanolamine; D012260:Ribonucleases; D019286:Endopeptidase K; D004047:Diethyl Pyrocarbonate