imaginal disc in situs

[Mary Stewart]

Hi,
Can anyone help us with advice on in situs to imaginal discs?  We made 
dig-labeled sense and antisense probes which worked great on embryos---no 
background with the sense probe.  We also have anti-dig antibody that was 
preabsorbed to embryos and this worked great on embryos.  When we tried 
these reagents on imaginal discs, we have a low, but unacceptable, level of 
background with the sense probe.  The antisense gives a stronger signal, 
but we're not comfortable with the results since we have some overall 
general staining with the sense probe as well as some staining that 
resembles what we see with the antisense (even though the antisense 
staining is more intense).  Does anyone have some advice on how we might 
solve these problems?  Also, any advice on how to get through all the 
handling steps and minimize the amount of tissue we lose?  We lost a lot of 
discs during all the handling and washing steps.  Thanks for any help.
Mary Stewart

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Mary Stewart
Assistant Professor
Department of Biological Sciences
North Dakota State University
Office:  701-231-8226
Lab:  701-231-6523