Hi Marc and Histonetters (especially those using XTs), I've been trying to work up VMS's new EBER probe with less than stellar results. I have some questions about the protocols you sent via Histonet back in early June. My questions/protocols are in red. You state the following: Depar 16 min We can only select "depar" without an incubation time....does your instrument allow you to select a time? We are doing this on an XT, is this an instrument difference? What instrument are you running your ISH on....Ultra by chance? Enzyme Protease 2 for 4 min. We are using ISH Protease 2 / 12 mins. as that was our protocol with the "old" stuff. ISH (EBER) probe 4 min. Same Denature @ 85 degrees for 12 min. Same Hybe 1 hour What temperature are you hybridizing at? 3 stringency washes for 8 minutes each At what temperature? Blue detection for 20 minutes Same Counterstain for 4 min We use Nuclear Fast Red for 8 mins. Marc, are you using HybReady? Anyone else having had some success with the new EBER probe, reagents and software, feel free to comment. Of the 3 specimens I've run, known positive soft tissue, bm bx and cell block, the soft tissue and bm bx had some positivity with the EBER DNP and U6 DNP but the cell block was negative with both. There was also lots of blue smearing and haze over the slides. I'm afraid this optimization/validation could end up being very expensive even with our 3 control tissues picked up together on single slides. Thanks for everyone's help, Linda
Linda A. Sebree University of Wisconsin Hospital & Clinics IHC/ISH Laboratory DB1-223 VAH 600 Highland Ave. Madison, WI 53792 (608)265-6596 _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet