[Drosophila] biotin-avidin-HRP background problem in doing FISH

Ying Wen wenvt5 at yahoo.com
Sat Dec 17 09:35:42 EST 2005


    Dear everone,
   
  I am a research asssitant working in Cincinnati Children's Hospital Research Foundation. I am currently doing dual-label FISH (Fluorecent Insitu Hybridization). I choosed the following schema:
   
  1.  dig labeld mRNA probe of gene1---------mouse anti-dig(Roche)---------Goat anti mouse with HRP(Molecular Biology)------Tyramide Signal Amplification (Molecular Biology)
   
  2. biotin labeld mRNA probe of gene2------------strepavidin-HRP (Jacson Immuno)------Tyramide Signal Amplification (Molecular Biology)
   
  The first one (dig) gave me nice staining, but the second one (strepavidin) always gave high background. I know the biotin labeled probe is good, since when I use this probe tested with mouse-anti-bio antibody, then followed by Goat-anti-mouse-HRP second antibody, it works beautifully. So, the problem could be biotin-avidin problem. 
   
  Did anyone experience same problem when doing FISH? Appreciate it!
   
  Sincerely Yours,
  Ying Wen
   


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