RAS Fish FISH: The Ultimate Guide to Fluorescent In Situ Hybridization

So, you've heard about FISH. It's that flashy acronym thrown around in labs that sounds more like a weekend hobby than a powerful molecular technique. Fluorescent In Situ Hybridization. It can feel intimidating, shrouded in complex protocols and expensive microscopes. But at its heart, FISH is beautifully simple: you make a fluorescently tagged piece of DNA (a probe) that finds and sticks to its matching sequence on a chromosome or inside a cell, making it glow under the right light. Let's ditch the jargon and talk about how you can actually make this work on your bench, today. First up, the probe. You don't always have to design and label your own from scratch. That's a rabbit hole for another day. For most of us starting out, the practical choice is buying a commercial probe specific for your target—like a gene, a chromosomal region, or a repetitive sequence. Companies provide them ready-to-use, with a fluorescent tag already attached. Your first real decision is what color? FITC (green), Cy3 (red), and DAPI (blue, a counterstain for all DNA) are the classic trio. Keep it simple to start. If you're trying to see if a specific gene is amplified in a tumor sample, a red probe for the gene and a green probe for a control region on the same chromosome is a solid, actionable plan. The probe arrives in a tiny tube. Handle it like precious treasure. It's light-sensitive and degrades with repeated freeze-thaw. Aliquot it immediately upon arrival into single-use portions, store them at minus twenty in a dark box, and never, ever let them sit on ice under bright lights. Now, let's get to the meat of it: your sample. Whether it's a metaphase chromosome spread from cultured cells or interphase nuclei from a tissue section, it has to be fixed. That usually means slides with cells fixed in a mix of methanol and acetic acid (for chromosomes) or formalin-fixed, paraffin-embedded (FFPE) tissue sections. For FFPE, the single most crucial step you will ever do is the pre-treatment. This isn't a place to rush. You have to peel away the formalin cross-links that are hiding your target DNA. Here's your actionable protocol: bake the slides at 60 degrees Celsius for an hour to melt the paraffin, then deparaffinize in xylene and ethanol series. Then, immerse the slides in a pre-warmed citrate-based antigen retrieval solution and microwave them. Not a gentle simmer—a proper boil for 15-20 minutes. Let it cool slowly in the solution for another 20. This heat-induced epitope retrieval is your best friend. It breaks those cross-links and makes the DNA accessible. After a quick wash, you need to digest away the proteins that are still gumming up the works. Pepsin or proteinase K. This step is a Goldilocks game. Too little, and your probe can't get in; too much, and you digest your sample into a ghostly haze. Start with the manufacturer's recommendation, but be prepared to titrate. Under the microscope, your tissue should look slightly peppered, not shredded. After digestion, you refix the sample briefly with formaldehyde to stabilize it, then dehydrate it through an ethanol series (70%, 90%, 100%) and let it air dry. Now, the magic moment: hybridization. You take your precious aliquot of probe, let it thaw in the dark, and mix it with the hybridization buffer. This buffer is a cocktail of salts and formamide that controls the stringency—how perfectly the probe must match its target to stick. A higher formamide concentration means you need a more perfect match, reducing background noise. Pipette the probe mix onto your sample, drop a coverslip, and seal the edges with rubber cement. This creates a tiny, humid chamber. Then, into the denaturation oven. You will co-denature both the probe and the sample DNA at around 73-80 degrees Celsius for a few minutes. This melts the double strands. Then, you drop the temperature to 37-42 degrees Celsius for the overnight hybridization. This is where the probe finds its home. The key here? A flat, accurate thermal cycler or a dedicated hybridization oven. Temperature wobbles here can ruin everything. Walk away, let it happen. Come back the next day. Time for the washes to remove any probe that's just lazily sticking to anything. These are the SSC washes, and their temperature and concentration are your second major stringency control. A typical post-hybridization wash might be in a solution of 0.4x SSC at 72 degrees Celsius, followed by a rinse in 2x SSC at room temperature. Be gentle but thorough. Keep the slides in the dark from here on out. The final step before looking is counterstaining. You'll mount the sample with an antifade mounting medium containing DAPI. DAPI stains all the DNA a lovely blue, giving you the context—the outline of the nucleus or the chromosomes. Put on a coverslip, let it set, and store it in the dark at four degrees Celsius. Now, the moment of truth: the microscope. You need an epifluorescence microscope with the right filter sets for your fluorophores. Find your sample using the DAPI channel first—everything will glow blue. Then, switch to the FITC or Cy3 filter. Don't gasp if you see a glowing haze at first. That's background. Look for specific, bright, punctate signals. For a gene amplification assay, you'll count distinct dots. A normal cell might have two red dots (one for each chromosome). An amplified cell might have a dozen little red dots or one giant bright red cluster. That's your answer. For a translocation probe, you'd see two different colored signals fused together. The real talk part? Things will go wrong. Your first few slides might be blank, or have overwhelming background, or the tissue might have washed away. It's okay. Troubleshooting is 50% of FISH. Blank slide? Check denaturation temperature—maybe the target DNA never opened up. Also, check your probe expiration date. High background? Increase the stringency of your washes. Use a hotter wash buffer or a lower salt concentration. Sample washed away? Your digestion was too harsh. Cut the time or concentration next time. The beauty of FISH is its visual directness. You're not looking at a graph or a number; you're seeing the genetic truth light up inside the very architecture of the cell. It's a powerful feeling. Start simple, be meticulous with your pre-treatment and temperatures, and don't be discouraged by initial failures. Every blank slide teaches you more about the delicate dance of DNA accessibility and probe binding. With these hands-on steps, you're not just reading about theory; you're ready to make something glow.