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●● CC2—Extended at 100 °C (HIER)

●● (ii) (ii) ISH—Protease 3 for 16 min (Enzyme Epitope

Retrieval).

●● Denaturation—20 min.

●● Hybridization—6 h.

●● Stringency Wash—72 °C.

●● SISH Multimer—32 min.

●● Silver Chromogen—4 min.

●● Red ISH Multimer—24 min.

●● Red Chromogen—8 min.

●● Counterstain—Hematoxylin II for 8 min; Bluing Reagent for

4 min.

FISH uses a fluorophore-labeled probe to identify the DNA

sequence of interest, in this case is HER2. Once hybridized to the

sequence of interest, the tissue section is viewed using a fluores-

cence microscope, similar to the bright field immunohistochemical

techniques. Either a single probe or dual probe cocktail can be

used to identify the HER2 DNA sequence and C17 centromere.

The concordance between Dual ISH and FISH in one study

was 98.5% with a specificity of 99% and a sensitivity of 96% [ 43 ].

The advantages of the bright field ISH technique over FISH tech-

nique include a more rapid turnaround time for reporting, the

ability to archive the stained slides (fluorescent signal of FISH

fades over time), and the cost of a fluorescence microscope when

compared to a conventional light microscope. Cell morphology is

also easier to access in a bright field ISH slide when compared with

3.3.2 Fluorescence
In Situ Hybridization (FISH)

Fig. 3 SISH: HER2 signals (black) are typically smaller and more discrete than

CEP 17 (red)

Duminda Subasinghe et al.