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Published online 2010 October 11. doi: 10.4103/1742-6413.70968

Table 1

Procedure for dual SISH and red ISH on cytological material on the BenchMark XT IHC/ISH staining machine

Day 1
 Fix slides in 4% neutral buffered formalin (NBF) for 2 h at the bench
 Wash slides in Tris buffered saline (TBS) 2×5 min
 Load slides in the staining machine and start the protocol for cytological material
 Cell conditioning: Incubate slides in mild followed by standard cell conditioning buffer (CC2) for 8 min each.
 After enzyme (protease 3) incubation for 8 min, warm up slides to 47°C prior to hybridization
 Incubate slides with HER-2 DNA probe and hybridize for 6 h at 47°C
 Warm up slides up to 67°C and incubate in SISH stringent wash buffer 3×8 min.
 Incubate slides with Red ISH-probe (Chr17 Probe) at 44oC for 4 min and further for 2 h.
 Wash slides in Red ISH stringent wash at 59°C for 3×8 min.
 Counterstain slides with hematoxylin II for 8 min followed by post counterstain with Bluing reagent for 4 min.
Day 2
 Wash slides in warm tap water for 10 min.
 Air dry slides completely (e.g. 15 min at 56°C)
 Coverslip with non-alcoholic/non-xylen based mounting medium