To begin, obtain the sample mounted slide after aging it. To pretreat the slide, dip it serially in coplin jars containing appropriate solutions for the specified time. Digest the tissue with 100 to 200 microliters of proteinase K digestion buffer, and incubate at room temperature for one minute.
To stop the digestion, immediately immerse the slide serially into ethanol solutions for two minutes each. Apply the FISH hybridization mix to the slide and cover the sample with a cover slip. Place the slides onto a hot plate, such as a Slide Moat hybridization system at 75 degrees Celsius for two to five minutes.
Then transfer the slide onto another hot plate set at 37 degrees Celsius to hybridize overnight. Next, dip the slide into the warm wash buffer and carefully remove the cover slip. Wash and stain the slide using appropriate reagents in the dark.
Quickly dip the slide into deionized water for no more than one second and place it on a paper towel. After drying, mount the slide with anti-fade mounting media. Place the cover slip and seal it with nail polish before imaging.
Using a 60X oil lens, capture the fluorescent signal in multiple Z-stacks. Finally, perform a maximum 3D projection to achieve the best resolution and apply deconvolution or other background clearing algorithms. In the triple negative breast cancer samples, nuclear FISH primarily shows two distinct dots per nucleus, representing her two ERBB2 signals.
In contrast, HER2-positive samples display abundant FISH signals, indicating different patterns of gene amplification. Additionally, some nuclei in HER2-positive samples may exhibit clusters, suggestive of extra chromosomal DNA hubs, which are focal points for increased gene amplification.
