Hi Donna, I had a time with this one as well. I use Dako's antibody on the Leica Bondmax. I run it at 1:50 using ER2 for 30 minutes. I would be careful going any stronger on the dilution, I got quite a bit of background with a 1:40, the 1:50 stains clean but can be a little weak if you don't use a fresh cut slide. I ultimately decided to go with the 1:50 because I could get cleaner, consistent staining with it.
An equivalent for this protocol would be an EDTA (High pH) retrieval in a pressure cooker for about 20 minutes with a 10 minute cool down. Antibody incubation on the Bond protocol is 15 minutes. I would recommend about 30 minutes on an open system or if you are doing it by hand. If you are running this on a Ventana platform I would start with a CC1 standard and at least 40 minutes on the antibody incubation. You may have to bump the retrieval time up to the extended. Good luck! Ashley Troutman BS, HT(ASCP) QIHC Immunohistochemistry Supervisor Vanderbilt University Histopathology 1301 Medical Center Drive TVC 4531 Nashville, TN 37232 _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
