Table 2 Nominal and optimization protocols for Dual in-situ hybridization (DISH)
From: Optimizing Ventana chromogenic dual in-situ hybridization for mucinous epithelial ovarian cancer
Selectable parameters | U1 (Nominal protocol) | U2 | U3 | U4 | U5 |
|---|---|---|---|---|---|
Year of samples | 2011 | 2008-2010 | 2003-2007 | 2000-2002 | 1990s |
Baking temperature | 63°C | 63°C | 63°C | 63°C | 63°C |
Baking time | 20 mins | 20 mins | 20 mins | 20 mins | 20 mins |
Deparaffinization | 72°C | 72°C | 72°C | 72°C | 72°C |
Extended deparaffinization | Not selected | Not selected | Not selected | Not selected | Not selected |
Cell conditioning duration | 3 cycles of CC2 at 86°C | 3 cycles of CC2 at 86°C | 3 cycles of CC2 at 86°C | 3 cycles of CC2 at 86°C | 3 cycles of CC2 at 86°C |
Mild CC2: 8 mins | Mild CC2: 8 mins | Mild CC2: 12 mins | Mild CC2: 16 mins | Mild CC2: 16 mins | |
Standard CC2: 12 mins | Standard CC2: 12 mins | Standard CC2: 12 mins | Standard CC2: 16 mins | Standard CC2: 16 mins | |
Extended CC2: 8 mins | Extended CC2: 8 mins | Extended CC2: 12 mins | Extended CC2: 16 mins | Extended CC2: 16 mins | |
ISH protease – 2 or 3, and duration | ISH protease 3 | ISH protease 2 | ISH protease 2 | ISH protease 2 | ISH protease2 |
-16 mins | -8 mins | -8 mins | -8 mins | -12 mins | |
Denaturation time | 20 mins | 20 mins | 20 mins | 20 mins | 20 mins |
Hybridization time | 6 hours | 6 hours | 6 hours | 6 hours | 6 hours |
Stringency wash temperature | 72°C | 72°C | 72°C | 72°C | 76°C |
SISH multimer incubation time | 32 mins | 36 mins | 36 mins | 36 mins | 36 mins |
Silver chromogen incubation time | 4 mins | 8 mins | 8 mins | 8 mins | 8 mins |
Red ISH multimer incubation time | 24 mins | 28 mins | 28 mins | 28 mins | 28 mins |
Red chromogen incubation time | 8 mins | 12 mins | 12 mins | 12 mins | 12 mins |
Hematoxylin II incubation time | 8 mins | 8 mins | 12 mins | 12 mins | 12 mins |
Bluing reagent incubation time | 4 mins | 4 mins | 8 mins | 8 mins | 8 mins |