Chromosomal aberrations are usually detected in most hematological and various solid tumors and, clinicopathologically, are frequently associated with the determination of immediate morphological and immunophenotypic features of the tumor. The detection of chromosomal aberrations provides the basis for line-by-line diagnosis and genetic classification of tumors. Especially in leukemias and lymphomas, many of the major chromosomal aberrations are associated with the clinical staging of the disease. In addition, other chromosomal aberrations will occur during tumor prognosis. Therefore, cytogenetic results are of great help in prognostic estimation and selection of therapeutic options.
Operation method
Fluorescence immunophenotyping and FISH co-analysis experiments on tumor cells
Materials and Instruments
Normal mouse serum Mouse monoclonal anti-digoxin antibody Mouse anti-FITC antibody AMCA-affinity FITC-affinity Biotinylated goat anti-affinity antibody Cy3-labeled affinity Cy3-labeled goat anti-mouse antibody Cy3-labeled rabbit anti-goat antibody Cy3-labeled donkey anti-rabbit antibody Cy3-labeled rabbit anti-rat antibody Digoxin-labeled goat anti-mouse anti-FITC-labeled donkey anti-mouse antibody FITC-labeled goat anti-digoxin antibody Buffer SSC Move I. Specimen preparation Simultaneous immunophenotyping and FISH can be performed using cell centrifugal preparation, smears, blots, or frozen sections. Proper handling and protection of cellular integrity are critical for successful completion of this part of the experiment. In general, freshly prepared specimen slices should be allowed to air dry overnight before transferring to -20°C or 1 80°C for storage. Specimens can be stored at low temperatures for weeks or even years. After removal from the refrigerator, specimens should be thawed at room temperature and allowed to dry before use. Before use, the cells should be examined under a phase contrast microscope for morphology and position. Here is how to make a centrifuged cell preparation. 1. Cut body tissues, such as lymph nodes or solid tumor tissues, into small pieces with scissors or a knife in PBS buffer. 2. Collect to obtain cell suspension. 3. Centrifuge at 200 g for IOmin. 4. Discard the supernatant and resuspend the cells in PBS. 5. Count the cells. 6. Adjust cell concentration to IXlO4 cells/mL. 7. Take 200, cell suspension into cell centrifuge, centrifuge at 200 g (800r/min) for 5 min. 8. Dry the specimen slice overnight or leave it at room temperature for at least 2 hours. 9. Store specimen slices at -20℃. Depending on the problem to be solved, one or two immunophenotyping experiments are performed. Here are two experimental methods. 16.70%, 85%, and 100% gradient ethanol dehydration for 3 min each, and air drying for at least lOmin in preparation for FISH experiments (see 3.3 above). Many multicopy probes have been commercialized, such as Vysis. general experiments use hybridization mastermixI, but with commercial probes it is recommended to follow the manufacturer's instructions. These probes are cloned on different vectors (from phage to BAC, PAC, YAC). after labeling of the DNA with biotin, digoxigenin, or synthetic dyes, the probes are centrifuged to reduce the reaction volume. Hybridization mastermixII can be used to solubilize the probe. Some commercial probes, such as coated probes, can also be used in this way. Probe preparation should be done according to the manufacturer's recommendations, e.g., for DNA probes purchased from Vysis, IfjtL Direct Labeling Probes should be added to 2pL of deionized water and 7pL of the supplied CEP Hybridization Buffer. We recommend using LifeTechnologies/Gibco's DNA Labeling Kit for Gap Shift Labeling in combination with biotin, digoxin, or a fluorescent dye such as green or orange. For each reaction, the IpgDNA probe is labeled at 15°C for lh. The labeled material is sampled on a Sephadex050 column and then centrifuged. 5% agarose gels are loaded with 5 |uL of the barbed shift-labeled probe, and the length of the probe fragments is examined, which should be 200?800 bp, with the majority being 300?600 bp. 1. Add the following reagents to an I.5 mL Eppendorf tube and allow to stand at -70°C for 30 min: 5-10ul of labeled DNA, 5ug of human Cot-1 DNA, human placenta DNA, 3ug of salmon sperm DNA, 3mol/L NaOAc (pH5.2) by 1/10 volume, and 2.5x the volume of 100% ethanol. Centrifuge at 13OOOg for 30 min at 2.4°C. 3. Dispose of the supernatant, add 2 times the volume of 70% ethanol to dissolve the remaining salt, and centrifuge at 13OOOg for 10 min. 4. Dry at room temperature. 5. 10uL hybridize mastermixI or mastermixII, mix well, and dissolve probe. If a single antigen immunophenotype is performed, a two-color HSH (biotin and digoxin probes) will be performed. In two-color FISH, equal amounts of digoxin and biotin probes are precipitated together and hybridized to the cells. When a dual immunophenotype is performed, a single FISH hybridization with the digoxin probe is sufficient (see Note 5). 1.2 to 3 uL of the hybridization mixture containing the DNA probe is added dropwise to the hybridization region. 2. Cover with a 12 mm cover slip. 3. Seal the piece with rubber cement and let it dry. 4. Place the specimen slice in the bottom of a metal box lined with a damp paper towel, cover the metal box and place it in a water bath at 76°C for 5 min (see Note 6). 5. Transfer the metal box to a 37°C incubator. Depending on the type of DNA probe, choose to hybridize for a few hours, overnight, or for several days. For repeat sequence probes, hybridization takes only 2 h, while for cDNA probes or chromosome coating probes, hybridization takes 2 days. 1. Remove the playdough. 2. Slide the specimen sheet gently off the cover sheet in preheated 0.1XSSC buffer (pH 7.0,60°C) (see Note 7). 3.0.1XSSC Wash the slides 3 times for 5 min each. 4. Soak the specimen slice in PN buffer for 3 min at room temperature. The following experimental method was used to detect the two-color probe (biotin and digoxin). When double immunophenotyping and FISH are performed simultaneously, only the digoxigenin probe is hybridized. The steps for detecting antibodies to biotin probes such as AMCA-affinity and biotin-labeled goat anti-affinity can be omitted (see Note 5). 1. Incubate a mixture of AMC-labeled affinity protein and monoclonal mouse anti-digoxin antibody (diluted 1:100 with PNM buffer, respectively) for 25 min at room temperature. 2. Wash the slices with PN buffer 3 times at room temperature for 5 min each time. 3. Incubate the mixture of biotin-labeled sheep anti-affinity antibody and digoxigenin-labeled sheep anti-mouse antibody (diluted 1:100 with PNM buffer, respectively) for 25 min at room temperature. 4. Wash the film with PN buffer 3 times at room temperature, 51^11 each time. 5. Incubate AMC-labeled affinity protein and FITC-labeled goat anti-digoxin antibody (diluted 1:100 with PNM buffer, respectively) for 25 min at room temperature. 6. Wash the film with PN buffer 3 times at room temperature for 5 min each time. 7. Seal the film with anti-quencher. If only red and green fluorescent dyes are used in the experiment, the specimen slides should be stained in DAPI solution for 2 min and rinsed at 2XSSC before sealing. Re-staining can help to determine the location of the hybridization signal in the nucleus. Immunophenotyping and FISH results can be viewed simultaneously or sequentially, depending on the monochrome, dichroic, or trichroic fluorescent filter system used. Results can be captured on high speed film (ASA400) or digital imaging systems. For immunophenotyping results, it is important to distinguish between true and false signals. Generally, the true signal is bright and fresh, while the false signal is faint or extremely bright.FISH analysis of cellular immunophenotypes can only be evaluated on intact cells with no overlap. We generally analyze a single specimen for at least 50 positive result cells and more than 100 negative result cells. It is important that the evaluation of the results be done by another independent observer. 1. For immunophenotyping experiments, monoclonal mouse anti-human CD antibody and polyclonal Cy3 and AMCA cross-linked antibody are diluted with PNM buffer at a ratio of 1:50?1:200. The appropriate antibody concentration is determined based on the antibody quality and the level of antigen expression in the cells. For example, CD3, which is highly expressed in T-lymphocytes, can be readily detected by incubation with only the first or first and second antibodies. A control experiment should be performed with each newly purchased antibody to determine its optimal concentration. In general, Cy3 and FITC are strong, reliable dyes, whereas AMCA is weak and unstable. results of Cy3 detection of immunophenotypes can be observed weeks or even months later, whereas the signal of AMCA is quickly quenched. specimen slices of FITC cannot be tested for more than a few days later because the autofluorescence of cells stains the background. 2. Re-fixation after immunophenotyping can be done with 1% or 4% paraformaldehyde. Paraformaldehyde can be stored at 4°C for more than 2 weeks, whereas Camoy fixative must be prepared fresh before each use. The duration of fixation in the buffer is very important for successful completion of the experiment. The shorter the cell fixation time, the more fluorescent signal is lost. However, too long a fixation time will result in a significant reduction in FISH hybridization efficiency. 3. In immunophenotyping, negative control experiments are necessary to exclude false negative results. Any fluorescent signal in the control experiment should be free of interference from heterogenous signals. Use monoclonal or primary antibodies in water, PBS, or unrelated isotype antibodies as negative controls. To exclude interference from interfering hybridization or polyclonal antibodies from different animal sources, control test slices should be subjected to double immunophenotyping experiments. 4. In double immunophenotyping experiments, incubation with 20% normal mouse serum containing a high concentration of murine immunoglobulin is essential for successful second immunophenotyping. The free site of the rabbit anti-mouse antibody is blocked and cannot bind to the second mouse anti-human antibody (e.g., CD8). No washes are required after this incubation. Double-immunophenotyped cells are FISH hybridized without biotin-labeled antibodies because they bind non-specifically to the second monoclonal mouse anti-human antibody. 5. Because of the limitation of colored fluorescence, it is difficult to combine three fluorescent colors in one experiment, such as green FITC, red Cy3, and blue AMCA.0 In fact, blue AMCA fluorescence is so weak and unstable that it can only be used for repeat sequence probes. On the other hand, FITC decreases the signal intensity faster than Cy3 and the contrast with the background will be reduced. Therefore, we recommend the use of Cy3 crosslinked antibodies to detect antigens with low expression or small position-specific DNA probes. 6. We did not find any difference in the results of denaturing cellular DNA and probes separately and simultaneously. Many laboratories use separate denaturation of target cell DNA and probe DNA, followed by pre-multiplexing of the DNA probe with human Cot-1DNA. We believe that simultaneous denaturation is quicker and easier, and the results are similar to separate denaturation. 7. In FISH studies, we have found that 3 hybridizations at 60°C in 0.IXSSC followed by elution are about as efficient as 50% formamide/2XSSC at 42°C, which is cheaper and non-toxic. For more product details, please visit Aladdin Scientific website.
Monoclonal mouse anti-human antibody Human Cot-1 DNA Human placental DNA Dextran sulfate Sephadex 050 Separation column Ultrasonically fragmented salmon sperm DNA 70%, 85%, and 100% Ethanol Rubbermaid Deionized formamide DAPI (4 6-diamino-2-phenylindole) DABCO (2 2 2-triethylene-1 4-diamine)
At this step the immunophenotyping results can be observed under a fluorescence microscope, the PN buffer is used to seal the slides, the glass coverslips are covered, and it is examined microscopically to see if a significant number of cells are fluorescing. If not, terminate the experiment and perform a control experiment with a different concentration of antibody. Note that the brightness of cell fluorescence is weaker when PN buffer is added to the specimen. Do not watch under the microscope for a long time.
