Basic experiments of FISH technique
Basic experiments of FISH technique
Source : Molecular Cytogenetics Techniques and Applications
Operation method
basic program
Materials and Instruments
70% ethanol SSC phosphate buffered solution (PBS) Hemo-De cleanup reagent Protease 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 tube rack in the center of the water bath so that it does not touch the edges of the water bath, and keep the water level just below the top of the tube rack throughout the procedure. 3. Adjust the concentration of the cell suspension with Carnoy's fixative, so that the suspension is mildly turbid. 4. Wash both sides of the slide with 70% ethanol and wipe the slide dry 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 over the water bath, take a 2~4in (1in=2.54cm) 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 a test tube rack in a water bath, with the specimen side up, and allow the slice to dry for 5 to 10 min. 8. Remove the slice and observe under a phase contrast microscope, adjusting the conditions of the drop if necessary. Second, the use of adjustable cytogenetic dryer to prepare medium-term chromosome specimens 1. Turn on the thermocouple vacuum gauge, set the temperature to 22℃ and relative humidity to 44%, and make it reach a stable state. 2. Adjust the cell concentration with Camoy fixative so that the suspension is slightly turbid. 3. Wash both sides of the slide with 70% ethanol and dry it with dust-free paper, and place the slice on the inner surface of the temperature-differential-electric-couple 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 suspension drying. The quality of the specimen prepared is generally better if the suspension dries within 45~60s. 6. Observe under the phase contrast microscope, and adjust the conditions of the drops if necessary. Preparation of uncultured specimens 1. Resuspend the fixed cells carefully. 2. Drop 15~20uL of cell suspension in each hybridization area (usually two areas per sheet) 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 in 2\SSC solution at 73°C for 2min. IV. Preparation of urethral epithelial cell specimens 1 Carefully resuspend the cells and drop 3uL, 10uL and 30uL of the suspension in 3 wells of a covered slide, respectively. 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 2min. V. Pre-treatment of fresh cells 1. soak the specimen in 2XSSC at 73℃ for 2min. 2. Soak the specimen in pepsin working solution at 37℃ for 10min 3. Wash the specimen in PBS at room temperature for 5min. 4. Place the specimen in post-treatment fixative for 5min at room temperature. 5. Wash the specimen in PBS at room temperature for 5min. 6. Dry the specimen naturally. 7. immersed the specimen in 70% ethanol for lmin at room temperature. 8. Soak the specimen in 85% ethanol for lmin at room temperature. 9. The specimen is immersed in 100% ethanol at room temperature for lmin. 10. Specimens can be denatured. Six, paraffin specimen pretreatment 1. Use a diamond pen to mark the paraffin area to be hybridized. 2. Dip 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 5min at room temperature. 5. Repeat step 4 with fresh ethanol. 6. Dry the specimen naturally or place the slice on a slice heating plate at 45~50°C for 2~5min. 7. Immerse the specimen in 0.2 mol/L HCl for 20min. 8. Immerse the specimen in pure water for 3min. 9. Immerse the specimen in elution buffer for 3min. 10. immersed the specimen in 80℃ pretreatment solution for 30min. 11. Immerse the specimen in purified water for 1min. 12. Immerse the specimen in elution buffer for 5min. 13. Repeat the washing step in elution buffer. 14. Dip a paper towel from the edge of the slide to remove excess elution buffer. 15. Dip the specimen in protease solution at 37°C for 10min. 16. Dip the specimen in elution buffer for 5min. 17. Repeat the washing step in elution buffer. 18. Dry the specimen on a specimen hot plate at 45~50°C for 2~5min. 19.Soak the specimen in neutral formalin buffer for 10min at room temperature. 20. Immerse the specimen in elution buffer for 5min. 21. Repeat the washing step in elution buffer. 22. Dry the specimen on a specimen hot plate at 45~50°C for 2~5min. 23. The hybridization procedure can be continued. VII. Hybridization and elution. Be sure to coordinate the time to prepare the probe and the time to denature the specimen so that they can be completed at the same time for hybridization. 1. soak the specimen in denaturing solution at 73°C for 5 min. denature up to 4~6 slides at a time in one vat. 2. Immerse the specimen in 70% ethanol at room temperature for lmin. 3. Immerse the specimen in 85% ethanol at room temperature for 1min. 4. Immerse the specimen in 100% ethanol at room temperature for 1min. 5. Pipet excess ethanol from the edge of the slide with absorbent paper 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 5min. 7. Add 3~10μL (depending on the sample area) of probe mix to the target area on the specimen. 8. Immediately cover the probe with a 22mmX22mm or a 12mm round coverslip to distribute the probe evenly. 9. Seal the perimeter of the coverslip with rubber cement. 10. Place the specimen in a 37°C preheated wet box. 11. Hold the hybridized specimens at 37℃ overnight (14~18h). 12. Gently remove the sealing adhesive with tweezers. 13. At room temperature, immerse the specimen in post-hybridization elution buffer and wash off the coverslip. 14. Remove excess liquid by pipetting from one end of the slide. 15. Dip the slide in post-hybridization elution buffer at 73°C for 2 min. No more than 4~6 non-formalin-fixed specimens should be eluted at a time per vat, and dip the slide in 2XSSC/0.1% NP-40 for 1 min at room temperature. 16. Remove the slice and place the slice vertically in a dark place to air dry. 17. Add 10jaLDAPI re-staining solution to the target area and cover with a 22mmX22mm coverslip. 18. After hybridization, store the specimen in the dark. Store at -20℃ when not in use. VIII. Signal counting of interphase cells Use each of the following criteria to assess the suitability of the slice. 1. Probe signal strength: The signal should be bright, clear and easy to assess. The signal should be tightly elliptical or linear and 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 40X eyepiece, scan a number of areas to estimate the heterogeneity of cell types. Choose an area with a uniform distribution of nuclei and avoid areas with weak target signals. 5. Using a 63X or 100X 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 in the nucleus. 7. Count two signals that are the same size and less than or equal to a single signal diameter apart. 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 of each color. Caveat 1. Although denaturation can be carried out by boiling the TE probe solution, some milder methods should be used in order to better preserve the morphology of tissues, cells and chromosomes. The degree of denaturation of DNA within chromatin also depends on a good understanding of the parameter: Tm value, defined as the temperature at which 50% of the DNA is in a single-stranded state. the magnitude of the Tm value depends on the GC content, monovalent cation concentration and double-strand length of the sample. the denaturation temperature must be well above the Tm value, so that nearly 100% of the DNA is in a single-stranded state. the typical denaturation conditions are 50% to 55% formamide/2X SSC at 70 to 75°C. the Tm is the temperature at which the sample is denatured. Typical denaturation conditions are at 70 to 75°C in 50% to 55% formamide/2X SSC solution. Typical denaturation conditions are 50% to 55% formamide/2X SSC solution at 70 to 75°C. Often probes and samples are denatured simultaneously on the slide, which provides more favorable kinetics for rapid formation of heteroduplexes. If the denaturation conditions are much higher than these conditions, extensive patchy hybridization patterns can be produced as a result of the de-helicalization of tight DNA-histone structures. Adequate immobilization prevents this to some extent. On the contrary, if the DNA strand is not denatured sufficiently, little or no hybridization signal will be produced. 2. The nature of the closed DNA varies depending on the complexity of the probe. Highly repetitive DNA probes such as mitotic or near-mitochondrial DNA must be hybridized in the presence of a 10- to 100-fold excess of total human genomic DNA. If the probe contains a single sequence from a specific region of a chromosome arm, it must be hybridized in the presence of a similar excess of highly repetitive DNA; this highly repetitive DNA, known as Cot-1 DNA, consists of highly repetitive a-sequences and moderately repetitive sequences (e.g., the Aki family of repetitions) The Alu repetitive family of sequences is present in all chromosomes and virtually all of the genomic DNA fragments used in the preparation of the probe. The Alu family of repeat sequences is present in all chromosomes and indeed in all genomic DNA fragments used to prepare probes. 3. The length of hybridization time is determined by the complexity of the probe sequence. Probes consisting of a significant portion of a single sequence usually require overnight hybridization at 37°C, whereas probes containing sequences of filaments or periplasmic satellites require only a few hours of hybridization at 37-42°C. The hybridization temperature can be varied slightly to distinguish highly related repeat sequences, although cross-hybridization can be effectively eliminated by high-tightness elution. Paraffin-embedded specimens can achieve similar specificity to non-paraffin-embedded specimens at higher stringency elution using a high-salt eluent, because the melting point of the probe to the target hybrid is lower in formalin-fixed specimens, possibly due to degradation that makes the target sequences too short or to the presence of proteins stabilized in the hybridization region that destabilize the hybrid. For more product details, please visit Aladdin Scientific website.
Water bath Humidifier Slides