Fluorescence in situ hybridization of multicolor fibers
Fluorescence in situ hybridization of multicolor fibers
Source : Molecular Cytogenetics Techniques and Applications
Operation method
Fluorescence in situ hybridization of multicolor fibers
Materials and Instruments
Cultured Cell Suspension PBS Halo Solution Bovine Serum Albumin Ethanol Ether 10 X Incision Buffer Nucleotide Mixture Bio-16-dUTP Dithiothreitol Yeast RNA Salmon Sperm DNA SSC TNT Solution TNB Solution Move I. Preparation of slide specimens containing DNA fibers Most types of cells are suitable for fiber FISH analysis as long as they remain unfixed or dry and the nuclei are intact. Frozen tissue specimens and precipitated cell clumps can still be used as long as the tissue can be resuspended into a cell suspension. Cells with large amounts of cytoplasm require more vigorous treatment to lyse them. It is common to start with the gentlest method and then increase the SDS treatment if lysis is not adequate. Place the following items on ice before preparation begins: 1. 0.05% SDS solution prepared from each halo solution and ultrapure water was placed in an IOOmL beaker and two other IOOmL beakers were filled with ultrapure water. 2. One test tube contains 20% BSA and the other contains PBS. 3. Glass plate (5 mm thick). Suitable for cultured cells, peripheral blood cells or other cell suspensions: 1. Load ImL cell suspension or trypsin-digested cells (10,000?100,000 cells) in Eppendor monitors and centrifuge for several seconds in a microcentrifuge. 2. Discard supernatant. 3. Resuspend cells in 20(^LPBS (4°C). For frozen tissues: 1. Cut one or more 45;xm thick slices of frozen tissue (see Note 3). 2. Place in an Eppendorf tube on dry ice. 3. Keep on dry ice until use. 4. Melt the tissue for about 30s. 5. Add about 50 VLPBS (4°C). 6. Vigorously pump the broken tissue pieces up and down until a suspension is formed. 1. Take 20pL of cells in a centrifuge tube, add PBS (4°C) to 47.5pL, add 2.5^20%BSA/PBS solution and mix well. 2. Take a drop of the above suspension onto the specimen and spread the liquid evenly over the whole slide with a pipette tip. 3. Place the slide on a cold glass plate for 2 min. 4. Stand the slide (frosted side up) vertically on a piece of paper to drain off excess liquid. 5. Air-dry the slide slowly with compressed air until the edges of the slide are dry (for easily lysed cells such as peripheral blood cells and cultured cells, steps 6?9 may be omitted) (see Note 4). 6. Gently immerse the slice vertically in Halo Solution 1 for 30s. 7. Slowly remove the sheet from the solution over a period of approximately 3s, and then over a period of 7s blot the excess liquid with paper (see step 4). 8. Gently immerse the sheet vertically in Halo Solution 2 for 30s. 9. Remove the sheet slowly for about 3s and then blot the excess liquid with paper for 7s (see step 4). 10. Gently immerse the sheet vertically in Halo Solution 3 for 45s. 11. Remove the sheet slowly for about 3s and then blot the excess liquid with paper for 7s (see step 4). 12. Wipe the back of the sheet with paper. 13. Place the sheet horizontally on a cold glass plate. 14. Place the wet sheet under a UV lamp at a distance of 1?IOcm (see Note 5). 15. Immediately irradiate for 1?IOmin at 254nm (see Note 6). 16. Place the slice vertically on the paper IOs (see step 4). 17. Gently immerse the sheet vertically in Halo Solution 4 for 30s. 18. Remove the sheet slowly for about 3s, then blot the excess liquid with paper for 7s (see step 4). 19. Gently immerse the sheet vertically in Halo Solution 5 for 30s. 20. Remove the sheet slowly over a period of about 3s and then blot the excess liquid with paper over a period of 7s (see step 4). 21. Wash twice with ultrapure water. 22. Stand the slice vertically on a rack to dry it and leave it at room temperature for a few minutes. 23. Observe the slides with a fluorescence microscope 16x objective and TRITC filter (see Note 6) and check for (a) moderate cell lysis (Fig. 1C) (see Note 7); (b) a sufficient number of cells (Fig. 1D) (see Note 8); and (c) a sufficient number of comet-like structures (i.e., fibers) (Fig. 1E) (see Note 8). 24. Allow to dry overnight at room temperature before hybridization; store in an airtight container at 20°C for long periods of time. 1. Prepare the following mixture in a centrifuge tube: 5 juL 10X Incision Buffer, 5^L 0.1m ○ l/LDTT, Know L Nucleic Acid Mixture, 0.5^L Labeled Nucleotides, ljugDNA, and adjust the volume to 44, with ultrapure water. 2. place the mixture on ice and add I1ULDNA polymerase. 3. Dilute the DNaseI stock solution with ice-cold ultrapure water in a 1:500/1:1000/1:2000 gradient. 4. Add 5pL of diluted DNaseI. 5. Incubate at 16°C for 2 h. 6. Place the reaction system on ice and take 5pL of the reaction system and test the fragment size on a 2% agarose gel (see Note 9). 7. Add 20.jugssDNA and 20jLig yeast RNA. 8. Add 0.1x volume of 3mol/LNaAc and 2.5x volume of anhydrous ethanol at 20°C to precipitate DNA. 9. Leave on ice-water mixture for 30 min. 10. Centrifuge at maximum speed for 30 min in a microcentrifuge to remove ethanol and air dry the precipitate. 11. Resuspend the probe in Hybridization Mix at a concentration of 30?60 ng/jaL, dissolve at 37°C for 30 min, and store away from light (4°C or 20°C). 1. Bake fiber-containing slides on a slide hotplate at 80°C for 2 h (see Note 10). 2. Prepare probe mixtures: 3 ng/VL of each labeled mucoid/PAC/P1 probe and 50-fold excess of Cot-1DNA for each probe and add hybridization mix to KVL. 3. Denature the probe mixture at 80°C for 8 min. 4. Anneal on ice for 2 min. 5. Mix well and slightly centrifuge in a microcentrifuge. 6. Pre-duplicate the probe in a water bath at 37°C for 30 min. 7. Add 12(^L denaturing mix (70% deionized formamide/2XSSC/50 mmol/L dextran sulfate) to a 24 mmX60 mm cover slip. 8. Place the fiber side of the slide over the cover slip to allow the denaturing mixture to spread naturally. 9. Turn the slide over so that the cover slip side is facing up and place the slide on a hot plate at 80°C for 3 min of precise heating. 10. Gently tilt the slide so that the cover slips off naturally. 11. Wash the slide with 2XSSC (4°C) on ice for 2 min. 12. Wash with 70% ethanol (20°C) for 5 min. 13. Dehydrate with 90% and 100% ethanol at room temperature. 14. Place the specimen vertically on a rack and air dry. 15. add IO^L of probe mixture to slide. 16. Cover with a 24 mmX24 mm cover slip. 17. Hybridize face down in a wet box at 37°C overnight. Preheat 400 mL of 2XSSC in a 37°C water bath. 1. Soak in a staining vat containing 2XSSC at 37°C for 5 min to allow the cover slip to slide off naturally (see Note 11). 2. Wash 3 times in 2XSSC at 37°C for 5 min each time. 3. Wash in TNT solution for 5 min. 4. Add 120 ^L TNB solution to a 24 mmX60 mm cover slip and place the fiber side of the slip over the cover slip. 5. Incubate face down for 20 min at 37°C in a wet box. 6. Prepare I:1000 murine anti-digoxin antibody and I:100 Texas Red-labeled streptavidin diluted in TNB solution at 12 ○ iuL per slide. 7. Elute the coverslips in TNT solution. 8. Add the antibody mixture to a 24 mmX60 mm coverslip and invert the slide to cover the coverslip. 9. Incubate face down for 20 min at 37°C in a wet box. 10. Elute the coverslip in TNT solution. 11. Wash 3 times in TNT solution for 5 min each time. 12. Prepare 1:1000FITC-labeled rabbit anti-mouse antibody and 1:100 biotinylated goat anti-streptavidin antibody in TNB solution, 12 ^L per slide. 13. The antibody mixture was added to 24 mmX60 mm cover slips and the inverted slides were covered to the cover slips. 14. Incubate face down for 30 min at 37°C in a wet box. 15. Prepare 1:100 FITC-labeled goat anti-rabbit antibody and 1:200 Texas Red-labeled streptavidin in TNB solution at 120/iL per slide. 16. Elute the coverslips in TNT solution. 17. Wash 3 times in TNT solution for 5 min each time. 18. Add the antibody mixture to a 24 mmX60 mm coverslip and invert the slide to cover the coverslip. 19. Incubate face down for 30 min at 37°C in a wet box. 20. Elute the coverslips in TNT solution. 21. Wash 3 times in TNT solution for 5 min each time. 22. Dehydrate with 70%, 90% and 100% ethanol series. 23. Air drying. 24. Add IO1^L of anti-quencher to the slide and cover with a 24 mmX60 mm cover slip. The very high background on the slide is due to the long fragmentation of the probe; however, this background is easily distinguishable from the linear arrangement of beaded fibers (Fig. 1E). FISH signals were analyzed according to the method of Vaandrager et al [11]. Known inter-probe distances and/or probe lengths (e.g. obtained by restriction enzyme mapping or pulsed-field gel electrophoresis) provide an internal length reference [14]. Differences in the degree of DNA condensation on different slides can be eliminated using computer-assisted extension and compression of digitized images to achieve the full length of a normal barcode. When abnormal barcodes, such as those caused by breaks, are present, the presence of a normal barcode signal from a normal allele confirms its hybridization efficiency. The number of full fibers necessary to locate a breakpoint is dependent on the number of tumor cells in the initial cell suspension. However, it is often possible to accurately localize breakpoints by measuring 5 normal versus abnormal barcode residues [14]. 1. Although many slide finishes are suitable for fiber FISH, in our experience special coating treatments are not necessary. 2. column-purified probe DNA is recommended for notch panning because DNaseI is very sensitive to contamination with very small amounts of phenol, salt, and RNA. 3. Specimens can be sliced and stored at 70°C for up to two months. The number of slices required per experiment depends on the conditions used to lyse the cells, and vigorous lysis can dislodge more cells from the slide. 4. Keeping the slide horizontal will result in an iridescent watermark. 5. UV irradiation creates gaps in the DNA. Too much irradiation will produce small DNA fragments that will fall off the slides, while too little irradiation will result in ineffective hybridization with little signal. Therefore, it is important to always determine the appropriate irradiation dose prior to each experiment, using irradiation time lengths that vary from 1?IOmin and distances between the UV lamp and the slide that vary from 1?IOcm. The Strata-Iinker device can also be used. It is important to remember that appropriate eye and skin protection is required throughout the UV irradiation. 6. Do not cover the coverslips and do not use oil. 7. A comet-like structure should appear (Fig. 1E). If the nuclei still appear as bright red globules, this indicates inadequate lysis (Fig. 1C). In this case, the following steps should be performed: slowly immerse the slide vertically in 0.05% SDS for 10?30s, drain off the excess liquid, immerse the slide in Halo Solution 1 for IOs and then continue with step 10. However, SDS often results in fewer cells being immobilized on the slide, and in this case, it is important to either use more cells or make the slide drier. The drier the slide, the more difficult the lysis will be, so care should be taken not to dry the specimen too much. 8. The number of fibers is sufficient when comet-like structures appear throughout the slide and when they are well aligned (Fig. 1E). If the slide is cloudy and bright crystalline material is visible, the number of cells is too high and should be reduced (Fig. 1D). If the entire slide is completely black (except for impurities), then the cell suspension should be examined after step 3 to confirm the presence of cells. If cells are easily observed in the suspension, it is possible that the cells were dislodged during the lysis step and the slide should be blown dry directly in step 6, skipping step 5, if possible completely drying the upper half, leaving the moist lower half, and then treated with Halo Solution 3. If the cells are still not lysed sufficiently after the above steps, the only option is to re-centrifuge the cell suspension, discard the supernatant, resuspend the cells in 50?IOOjULFCS and freeze overnight at 70°C, then thaw quickly and repeat the experiment from step 3.I.1. 9. The probe is labeled with biotin and/or digoxin, and the ideal probe fragment size is 400-800 bp. The size of the probe fragment can be controlled by adjusting the amount of DNaseI using a standard notch-panning procedure. The fragment size can be checked by 2% agarose gel electrophoresis with germ cell fibers. After labeling, the probes are precipitated and dissolved in the hybridization mix, allowing multiple probes to be mixed simultaneously without altering the hybridization conditions. 10. Slides that have been dried overnight at room temperature or stored at 20°C can be used. 11. Do not use external force to remove coverslips at any time; they should be dislodged naturally by immersion in a staining vat. For more product details, please visit Aladdin Scientific website.
Microcentrifuge Camag UV Box II Fluorescence Microscope Probe DNA