Experimental principle The basic principle of FISH is to use a known labeled single-stranded nucleic acid as a probe, according to the principle of base complementarity, heterosexually bind to an unknown single-stranded nucleic acid in the material to be tested to form a hybrid double-stranded nucleic acid that can be detected. Since DNA molecules are arranged linearly on the chromosome along the longitudinal axis of the chromosome, the probe can directly hybridize with the chromosome to locate the specific gene on the chromosome. Compared with traditional radiolabeled in situ hybridization, fluorescent in situ hybridization has the characteristics of rapid, strong detection signal, high hybridization specificity and multiple staining, so it has received widespread attention in the field of molecular cytogenetics. The probes used for hybridization can be roughly classified into three categories: 1. Chromosome-specific repeat sequence probes, such as alpha satellites and satellite III probes, whose hybridization target is often greater than 1Mb, do not contain interspersed repeats, and bind tightly to the target, strong hybridization signal, easy to detect; 2. Whole chromosome or chromosome region-specific probes, which are composed of extremely different nucleotide fragments on a chromosome or a certain segment of a chromosome, and can be obtained from chromosome-specific large fragments cloned into phages and plasmids; 3. Specific position probes, consisting of one or several cloned sequences. The fluorescein labeling of the probe can use direct and indirect labeling methods. Indirect labeling is the use of biotin-labeled DNA probes. After hybridization, lotus root is linked to fluorescein or streptavidin for detection. At the same time, the avidin-biotin-fluorescein complex can be used to convert the fluorescent signal. Amplify so that 500bp fragments can be detected. In the direct labeling method, fluorescein is directly covalently bound to the probe nucleotide or pentose phosphate backbone, or the fluorescein nucleoside triphosphate is incorporated when the probe is labeled by the gap translation method. The direct labeling method has simple steps during detection, but because signal amplification cannot be performed, the sensitivity is not as good as the indirect labeling method. Experimental equipment and reagents (1) Experimental equipment Constant temperature water bath, incubator, staining tank, Nikon E400 fluorescence microscope (2) Reagents Human MyoD1 (MYF3) gene probe, human peripheral blood metaphase chromosome cell specimen, nail polish, formamide, potassium chloride, sodium citrate, sodium hydroxide, Tween 20 Experimental procedure (1) Preparation of solution 1. 20 × SSC: 175.3g NaCl, 88.2g sodium citrate, add water to 1000mL (adjust pH to 7.0 with 10mol / L NaOH). 2. Deionized formamide (DF): Add 10g mixed bed ion exchange resin to 100mL formamide. Stir electromagnetically for 30 min and filter with Whatman No. 1 filter paper. 3. Volume fraction 70% formamide / 2 × SSC: 35 mL formamide, 5 mL 20 × SSC, 10 mL water. 4. Volume fraction 50% formamide / 2 × SSC: 100 mL formamide, 20 mL 20 × SSC, 80 mL water. 5. Volume fraction 50% dextran sulfate (DS): melted in 65oC water bath, stored at 4oC or -20oC. 6. Hybridization solution: 8 μL volume fraction 25% DS, 20 μL 20 × SSC mixed. (Or 40μL volume fraction 50% DS, 20μL 20 × SSC, 40μL ddH2O mix) Take 50μL of the above mixture and mix it with 5μL DF. The final concentration is 10% DS by volume, 2 × SSC, and 50% DF by volume. 7. PI / antifade solution PI stock solution: first dissolve the solution in double distilled water, the concentration is 100 μg / mL, take out 1 mL, add 39 mL of double distilled water to make the final concentration 2.5 μg / mL. Antifade stock solution: Prepare this solution with PBS buffer to make its concentration 10mg / mL, and adjust the pH value to 8.0 with 0.5mmol / L NaHCO3. Take 1 mL of the above solution, add 9 mL of glycerin, and mix well. PI / antifade solution: PI and antifade stock solution should be mixed well according to the volume ratio of 1: 9, and stored at -20oC for future use. 8. DAPI / antifade solution: prepare 1mL / mg DAPI stock solution with deionized water, dilute the antifade solution into working solution according to the volume ratio of 1: 300. 9. Blocking solution I: volume fraction 5% BSA 3mL, 20 × SSC 1mL, ddH2O 1mL, Tween20 5μL mixed. 10. Blocking solution II: volume fraction 5% BSA 3mL, 20 × SSC 1mL, goat serum 250μL, ddH2O 750μL, Tween20 5μL mixed. 11. Fluorescence detection reagent diluent: volume fraction 5% BSA 1mL, 20 × SSC 1mL, ddH2O 3mL, Tween20 5μL mixed. 12. Eluent: 100mL 20 × SSC, add water to 500mL, add 500μL of Tween20. (2) Operation steps Probe denaturation Incubate the probe in a 75oC constant temperature water bath for 5 minutes, immediately set it to 0oC for 5 to 10 minutes to denature the double-stranded DNA probe. 2. Specimen degeneration 1) Place the prepared chromosome slide specimens in a 50oC incubator and bake them for 2 ~ 3h. (Specimens stained by Giemsa need to be faded in fixative before baking.) 2) Take out the slide specimen and immerse it in 70-75oC volume fraction of 70% formamide / 2 × SSC denaturation solution for denaturation for 2-3 minutes. 3) Immediately dehydrate the specimens in a sequence of 70% volume fraction, 90% volume fraction, and 100% volume fraction ice-cold ethanol for 5 minutes each time, and then air dry. 3. Hybridization Drop 10μL of denatured or pre-annealed DNA probe onto the denatured and dehydrated slide specimen, cover with 18 × 18 coverslip, seal with Parafilm, and place in a wet cassette at 37oC for hybridization overnight (about 15-17h ). Because there is less hybridization solution, and the hybridization temperature is higher and the duration is longer, this process is carried out in a wet box in order to maintain the wet state of the specimen. 4. Elution This step helps to remove non-specifically bound probes, thereby reducing background. 1) The next day after hybridization, remove the specimen from the 37oC incubator, and gently remove the cover slip with a blade. 2) Place the hybridized slide specimens in preheated volume fraction of 50% formamide / 2 × SSC at 42-50 ° C and wash 3 times for 5 minutes each time. 3) Wash 3 times in 1 × SSC that has been preheated at 42-50oC for 5 minutes each time. 4) At room temperature, gently wash the slide specimens in 2 × SSC. 5) Remove the slide and let it dry naturally. 6) Take 200 μL of counterstain solution (PI / antifade or DAPI / antifade staining solution) dropwise on the slide specimen and cover with a coverslip. 5. Amplification of hybridization signal (applicable to probes labeled with biotin) 1) Add 150 μL of blocking solution I to the hybridization site of the slide, cover with plastic wrap, and incubate at 37 ° C for 20 min. 2) Remove the plastic wrap, add 150 μL of avidin-FITC to the specimen, cover with plastic wrap, and continue to incubate at 37oC for 40 minutes. 3) Take out the specimen, put it in the pre-heated 42-50oC eluent and wash 3 times, 5min each time. 4) Add 150 μL of blocking solution II to the hybridization site of the slide specimen, cover with plastic wrap, and incubate at 37oC for 20 min. 5) Remove the cling film, add 150μL antiavidin to the specimen, cover the new cling film, and incubate at 37oC for 40min. 6) Take out the specimen, put it into the new eluent which has been preheated at 42-50oC, and wash it 3 times, 5min each time. 7) Repeat steps (1), (2), (3), and then wash in 2 × SSC at room temperature. 8) Remove the slide and let it dry naturally. 9) Take 200μL of PI / antifade dye drop on the slide specimen and cover with a coverslip. 6. Sealing Different types of mounting fluids can be used. If the mounting fluid does not contain Mowiol (which can cause the sealing fluid to self-sealing), in order to prevent the solution between the coverslip and the slide from volatilizing, use nail polish to seal the coverslip. The sealed slide specimen can be kept in the cassette in the refrigerator at -20 to -70oC for several months. 7. Observe the results of FISH with a fluorescence microscope First find the field of view with cell division phase under the visible light source, then turn on the fluorescent excitation light source, the excitation wavelength of FITC is 490nm. The cells were stained red by PI, and the FITC-labeled probes fluoresced green. Shanghai Canspec Scientific Instruments Co., Ltd. is a intensive supplier dedicated to a full set of laboratory supplies and equipment (including: biology, chemistry, chemicals, laboratory consumables, instruments, etc.) Provide experimental products for domestic industries. 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