[Histonet] PNA Telomere Probe

From:Swaram


   Hello Histonetters,
   I  am  using Cy3-PNA Telomere Probe from ABI on 8-10 year old paraffin
   fixed  tissues.  I also have a Cy5-Centromere probe in the same mix. I
   deparaffinize,  treat  with NaSCN 80C (4min), Pepsin 37C (8min), air
   dry and then denature in denaturing solution 74C, air dry again, keep
   sections  on heating block 37C and apply 10l of the Probe mix, cover
   with a small piece of plastic coverslip, heat to 81C in a Hybrite and
   then  leave  OVT at 37C in a humidity chamber. Washing is done in 50%
   CH3-NO,  2X  SSC  and  2X  SSC-Tween-20, mounted with DAPI and Prolong
   antifade.
   However, I get good signals of the Cy5-centromere probe, but mostly no
   signals of the Telomere Probe. Sometimes, in the same tissue I get the
   Telomere  probe  signals (faint) but then I also see some non specific
   signals  in the cytoplasm. I have run controls using metaphase spreads
   and  verified  that  the Cy3-Telomere probe works fine. Please help me
   overcome   the  high  background  and  non  specific  signals  in  the
   Cy3-Telomere  PNA  Probe.  Interestingly,  the  first time, I ran this
   experiment,  I  got really good signals on both Cy3-Cy5. But I started
   getting problems after I changed the lot of the probe.
   Sincerely,
   Swaram
   Univ. California
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