FISH Basic Techniques and Problem Solving
FISH Basic Techniques and Problem Solving
Fluorescence in situ hybridization (FISH) technology allows the detection of specific nucleic acid sequences in chromosomes, cells and tissues. The unambiguous detection of altered structure or copy number of a whole chromosome or a specific region of a chromosome is an important predictor of human disease. FISH for chromosome analysis can be divided into intermediate FISH and interphase FISH, with the main difference between the two being in the preparation of cells prior to pretreatment and hybridization. Cell culture methods for interphase analysis are the same as those used for standard karyotyping, and FISH is most effective in specimens with few cellular structures or containing cells of different populations.The FISH technique is applicable to a wide range of specimen types, particularly archival specimens such as formalin-fixed, paraffin-embedded tissues.The FISH technique can be applied to a wide range of specimen types, especially archival specimens. However, the success of probe hybridization depends on the preparation of the specimen. In interphase FISH, pretreatment with a variety of reagents that increase the permeability of target cells or tissues but maintain the morphologic structure of important tissues greatly improves the hybridization efficiency of the probe. In addition, pretreatment reduces the spontaneous incandescent background of tissues or cells. A thorough understanding of the principles of the specimen pretreatment process can help solve problems that arise during the experiment. Specimen DNA-dan can be used for hybridization, and the experimental conditions for obtaining the most optimal signal should generally not be altered. The purpose of this chapter is to describe the salient features of FISH with respect to specimen preparation and hybridization.
Operation method
FISH Basic Techniques and Problem Solving
Materials and Instruments
Carnoy fixative Ethanol Cell suspension 2 X SSC pre-warmed pepsin working solution Phosphate buffer solution Denaturing solution Post hybridization eluent DAPI recombinant staining solution Move I. Preparation of medium-term cell specimens for culture 1. Place a water bath and a humidifier in a small separate room. The humidity should be approximately 50%; if the room hygrometer indicates less than 45%, a humidifier should be used. 2. Preheat the water bath to 67°C ± 2°C. Place a test tube rack in the center of the water bath so that it does not touch the edge of the water bath and keep the water level just below the top of the rack throughout the procedure. 3. Adjust the concentration of the cell suspension with Carnoy's Fixative so that the suspension is slightly cloudy (see Note 3). 4. Wash both sides of the slide with 70% ethanol and dry the slide with dust-free paper. 5. Immerse the cleaned slide in a vat of water and tilt the slide so that the water evenly covers the upper surface of the slides. 6. Immediately move the slide to the top of the water bath and take a 2-4in (lin=2.54 cm) Pasteur pipette and place 3 or 4 drops of cell suspension on the top of the slide along the long side of the slide. 7. Place the slice on the top of the test tube holder in the water bath with the specimen side up and allow the slice to dry for 5~IOmin08. Remove the slice and observe it under a phase contrast microscope, adjusting the conditions of the drops as necessary. 1. Turn on the thermocouple vacuum gauge and set the temperature to 22°C and relative humidity to 44% and allow to stabilize. 2. Adjust the cell concentration with Camoy Fixative so that the suspension is slightly turbid (see Note 5). 3. Wash both sides of the slide with 70% ethanol and dry with dust-free paper, and place the slides on the inner surface of a temperature-differential coupling vacuum gauge. 4. Holding a 2~4in Pasteur pipette, place 3 or 4 drops of cell suspension along the long side of the slide directly above the slide. 5. Observe the drying of the suspension. Specimens prepared when the suspension dries within 45-60 seconds are generally of good quality. 6. Observe under a phase contrast microscope and adjust the conditions of the slide if necessary. 1. Carefully resuspend fixed cells. 2. Drop 15~20uL of cell suspension per hybridization region (usually two regions per slice) 3. Air-dry the specimen. 4. Examine cell density under a phase contrast microscope and repeat steps 2 and 3 if necessary. 5. Age overnight or hold at 73°(: in 2\83(: solution for 21^11 (see Note 7). 1. Carefully resuspend the cells and drop 3ul, 10uL and 30uL of suspension in 3 wells of covered slides. 2. Air dry the specimen. 3. determine which sample has the best cell density, i.e., has a sufficient number of non-overlapping cells. 4. observe the cell density under a phase contrast microscope and select the appropriate wells for hybridization. 5. Age overnight or hold in 2XSSC solution at 73°C for 2 min (see Note 7). 1. Soak the specimen in 2XSSC at 73°C for 2 min. 2. Soak the specimen in pepsin working solution at 37°C for lOmin. 3. Wash the specimen in PBS at room temperature for 5 min. 4. Leave the specimen in the post-treatment fixative for 5 min at room temperature. 5. Wash the specimen in PBS at room temperature for 5 min. 6. Dry the specimen naturally. 7. Immerse the specimen in 70% ethanol for lmin at room temperature. 8. Immerse the specimen in 85% ethanol for 1 min at room temperature. 9. Immerse the specimen in 100% ethanol for 1 min at room temperature. 10. The specimen can be denatured. 1. Mark the area of paraffin to be hybridized with a diamond pen. 2. Immerse the specimen in Hemo-De Cleaning Reagent for lOmin at room temperature. 3. Repeat washing the specimen in Hemo-De Cleanup Reagent 2 times, using fresh Hemo-De Cleanup Reagent each time. 4. Dehydrate the specimen in 100% ethanol for 5 minutes at room temperature. 5. Repeat step 4 with new ethanol. 6. Allow the specimen to dry naturally or place the slice on a slice heating plate at 45?50°C for 2?5 min. 7. Immerse the specimen in 0.2 mol/LHCl for 20 min (see Note 9). 8. Immerse the specimen in purified water for 3 min. 9. Immerse the specimen in elution buffer for 3 min. 10. Immerse the specimen in 80°C pretreatment solution for 30 min. 11. Immerse the specimen in purified water for 1 min. 12. Immerse the specimen in elution buffer for 5 min. 13. Repeat the washing procedure in elution buffer. 14. Dip a paper towel from the edge of the slide to remove excess Elution Buffer. 15. Immerse the specimen in protease solution at 37°C for IOmin (see Note 10). 16. Immerse the specimen in elution buffer for 5 min. 17. Repeat the washing step in elution buffer. 18. Dry the specimen on a specimen hot plate at 45~50°C for 2~5 min. 19. Soak specimens in neutral formalin buffer for IOmin at room temperature (see Note 11). 20. Immerse the specimen in elution buffer for 5 min. 21. Repeat the washing step in elution buffer. 22. Dry the specimen on a specimen hot plate at 45?50°C for 2?5 min. 23. The hybridization procedure can be continued. Be sure to coordinate the time to prepare the probe and the time to denature the specimen so that they are done at the same time in preparation for hybridization. 1. soak specimens in denaturing solution at 73°C for 5 min. denature up to 4?6 slides at a time in one vat (see Note 12). 2. Immerse the specimen in 70% ethanol for 1 min at room temperature. 3. Immerse the specimen in 85% ethanol at room temperature for 1 minute. 4. Immerse the specimen in 100% ethanol at room temperature for 1 minute. 5. Use absorbent paper to draw excess ethanol from the edge of the slide and dry the back of the slide with a paper towel. 6. Dispense the probe into a microcentrifuge tube and cap tightly, denature the probe in a 73°C water bath for 5 min. 7. Add 3 to 10 uL (depending on sample area) of Probe Mix (see Note 13) to the target area on the specimen. 8. Immediately cover the probe with a 22 mmX22 mm or a 12 mm circular (3.4 Urologic Epithelial Cell Hybridization in this chapter) coverslip to distribute the probe evenly. 9. Seal the perimeter of the coverslip with rubber cement. 10. place the specimen in a preheated wet box at 37°C. 11. Hold hybridized specimens at 37°C overnight (14?18 h, see Note 14). 12. Gently remove the sealing adhesive with forceps. 13. At room temperature, immerse the specimen in post-hybridization elution buffer and wash off the coverslip. 14. aspirate the excess liquid from one end of the slide. 15. Dip slides in Post-Hybridization Elution Buffer at 73°C for 2 min. No more than 4?6 non-formalin-fixed specimens per vat should be eluted at a time, and Imin in 2XSSC/0.1% NP-40 at room temperature (see Note 14). 16. Remove the slides and place them vertically in a dark place to air dry. 17. Add IOjaLDAPI Restaining Solution to the target area and cover with a 22 mmX22 mm coverslip. 18. After hybridization, store the specimens in the dark. When not in use, store at 1 20°C. Use each of the following criteria to assess the appropriateness of the slice (see Notes 15 to 20). 1. Probe signal strength: The signal should be bright, clear, and easy to assess. The signal should be tightly elliptical or linear, diffuse elliptical. 2. Background: The background should be dark or black with few fluorescent particles or haze... 3. Identification of the target signal: Use the specified filter. Adjust the focus and familiarize yourself with the size and shape of the target signal and background noise (debris). 4. Using a 40 mesh eyepiece, scan a number of areas to estimate cell type heterogeneity. Choose an area with a uniform distribution of nuclei and avoid areas with weak target signals. 5. Using a 63X or IOOX eyepiece, begin analyzing the upper left quadrant of the selected area and scan from left to right, following the guidelines for counting signals within the boundaries of the nuclei of each clearly identified interphase cell. 6. Adjust the focus up and down to find all signals within the nucleus. 7. Same size, less than or equal to the diameter of a single signal can be counted as two signals. 8. Do not count nuclei that have no signal or that have only one color signal when two or more different fluoresceins are used. Only count nuclei that have one or more FISH signals per color. For more product details, please visit Aladdin Scientific website.
Water bath Humidifier Plastic wrap Hygrometer Test tube racks Dust free paper Phase contrast microscope Drying box DNA probes Tweezers Hybridization wet box warmer