33P is preferable to 35S for labeling probes used in in situ hybridization.
We compared RNA probes labeled with 35S-UTP and 33P-UTP for use in in situ hybridizations. 33P-UTP was readily incorporated into in vitro transcribed RNA, producing 33P-labeled riboprobes of high specific activity. When the 33P- and 35S-labeled riboprobes were compared in in situ hybridizations using two different tissues, we found that the 33P-labeled riboprobes were less "sticky" than the 35S-labeled riboprobes, giving significantly less nonspecific background hybridization. Because of the low level of background stickiness, it was possible to use ten times more 33P-labeled riboprobe than 35S-labeled riboprobe without appreciably increasing background hybridization. Our findings indicate that, in most cases, 33P is the isotope of choice when labeling probes for in situ hybridizations.
McLaughlin
S K
SK
Roche Research Center, Roche Institute of Molecular Biology, Nutley, NJ 07110.
Margolskee
R F
RF
D000818:Animals; D017403:In Situ Hybridization; D007553:Isotope Labeling; D010761:Phosphorus Radioisotopes; D015347:RNA Probes; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D012160:Retina / Q000737:chemistry; D013462:Sulfur Radioisotopes; D013650:Taste Buds / Q000737:chemistry; D014158:Transcription, Genetic; D015293:Transducin / Q000235:genetics; D014544:Uridine Triphosphate / Q000378:metabolism
D010761:Phosphorus Radioisotopes; D015347:RNA Probes; D012333:RNA, Messenger; D013462:Sulfur Radioisotopes; C075192:gustducin; D015293:Transducin; D014544:Uridine Triphosphate