Table 3 Individualized optimization (U6): protocol used for optimization of a 2003 case and comparison with first DISH protocol U3
From: Optimizing Ventana chromogenic dual in-situ hybridization for mucinous epithelial ovarian cancer
Selectable parameters | Protocol used for first DISH analysis (U3) | Protocol used for second DISH analysis (U6) |
|---|---|---|
Baking temperature | 63°C | 63°C |
Baking time | 20 mins | 20 mins |
Deparaffinization | 72°C | 72°C |
Extended deparaffinization | Not selected | Not selected |
Cell conditioning duration | 3 cycles of cell conditioning 2 (CC2) at 86°C | 3 cycles of cell conditioning 2 (CC2) at 86°C |
Mild CC2: 12 mins | Mild CC2: 16 mins | |
Standard CC2: 12 mins | Standard CC2: 16 mins | |
Extended CC2: 12 mins | Extended CC2: 16 mins | |
ISH protease – 2 or 3, and duration | ISH protease 2 – 8 mins | ISH protease 2 – 12 mins |
Denaturation time | 20 mins | 20 minutes |
Hybridization time | 6 hours | 6 hours |
Stringency wash temperature | 72°C | 72°C |
SISH multimer incubation time | 36 mins | 32 mins |
Silver chromogen incubation time | 8 mins | 4 mins |
Red ISH multimer incubation time | 28 mins | 24 mins |
Red chromogen incubation time | 12 mins | 8 mins |
Hematoxylin II incubation time | 12 mins | 8 mins |
Bluing reagent incubation time | 8 mins | 4 mins |