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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
  1. The incubation lengths of various selectable parameters that were changed are in bold.
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