Whole mount in situ detection of low abundance transcripts of the myogenic factor qmf1 and myosin heavy chain protein in quail embryos.
We describe a whole mount in situ hybridization procedure to detect and localize low abundance transcripts (qmf1) and myosin heavy chain proteins in quail embryos. The critical factor in transcript detection was the extent of proteinase K digestion. Optimal digestion increased probe accessibility and reduced background. The use of digoxigenin-labeled probes and immunological detection with anti-digoxigenin-alkaline phosphatase conjugated antibody allowed signal development in 6-10 h, unlike the 7 days required using 35S-labeled probes. Myosin heavy chain proteins were detected using immunofluorescence and confocal laser scanning microscopy to allow precise localization of signal within developing embryos.
Coutinho
L L
LL
Department of Genetics, University of Georgia, Athens 30602.
Morris
J
J
Ivarie
R
R
D000818:Animals; D003370:Coturnix; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D009124:Muscle Proteins / Q000032:analysis / Q000235:genetics*; D009218:Myosins / Q000032:analysis / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D013048:Specimen Handling
D009124:Muscle Proteins; D012333:RNA, Messenger; D009218:Myosins