Ventana discovery xt instrument

Example 15

This example discusses staining for pAKT on Calu-3 Xenografts. In this example, freshly harvested Calu-3 xenograft tumors were sliced into two roughly equal portions and treated with either 10% NBF (Formalin) at RT or 10% NBF at 4 degrees C. Tumor samples treated with RT Formalin were placed directly into the solution within 5 minutes of tumor removal. Samples were then removed from the formalin at time intervals of 0, 2, 4 or 24 hours after submersion. One tumor sample was placed directly into formalin at 4° C. for 2 hours and then transferred to 45° C. formalin for an additional 2 hours. All samples were placed into 70% ethanol as a holding reservoir until being processed further in an automated tissue processor set to an overnight program. All samples were embedded into paraffin wax for microtomy. Slides were stained with an antibody recognizing pAKT epitope (Cell Signaling Technologies, Cat #4060) on an automated slide stainer (VENTANA Discovery XT instrument, Ventana Medical Systems, Inc.). As shown in FIGS. 11A-E, significantly more pAKT was retained in the present dual temperature fix (2+2) protocol than standard RT formalin processes (compare 24 hour to 2+2 panels).

Example 15

This example discusses staining for pAKT on Calu-3 Xenografts. In this example, freshly harvested Calu-3 xenograft tumors were sliced into two roughly equal portions and treated with either 10% NBF (Formalin) at RT or 10% NBF at 4 degrees C. Tumor samples treated with RT Formalin were placed directly into the solution within 5 minutes of tumor removal. Samples were then removed from the formalin at time intervals of 0, 2, 4 or 24 hours after submersion. One tumor sample was placed directly into formalin at 4° C. for 2 hours and then transferred to 45° C. formalin for an additional 2 hours. All samples were placed into 70% ethanol as a holding reservoir until being processed further in an automated tissue processor set to an overnight program. All samples were embedded into paraffin wax for microtomy. Slides were stained with an antibody recognizing pAKT epitope (Cell Signaling Technologies, Cat#4060) on an automated slide stainer (VENTANA Discovery XT instrument, Ventana Medical Systems, Inc.). As shown in FIGS. 11A-E, significantly more pAKT was retained in the present dual temperature fix (2+2) protocol than standard RT formalin processes (compare 24 hour to 2+2 panels).