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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
  1. CC2: Cell Conditioning 2. Parameters highlighted in bold indicate changes from nominal protocol U1.
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