[Histonet] PNA Telomere Probe

[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 80°C (4min), Pepsin 37°C (8min), air
   dry and then denature in denaturing solution 74°C, air dry again, keep
   sections  on heating block 37°C and apply 10µl of the Probe mix, cover
   with a small piece of plastic coverslip, heat to 81°C in a Hybrite and
   then  leave  OVT at 37°C 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