Color Tape Experiment

Summary

The interspecies color banding technique is a simple and rapid method for detecting chromosomal abnormalities that cannot be identified by G-banding. This technique is particularly useful when GTG bands do not provide sufficient clues for single chromosome smear probe analysis. In addition, color banding can detect intrachromosomal abnormalities such as inversions, duplications, and small deletions that cannot be detected by other multiplex FISH techniques (e.g., M-HSH and SKY).

Operation method

Color Tape Experiment

Materials and Instruments

Chromosome specimens prepared by conventional cytogenetic methods
RxFISH probe Formamide Ethanol Methanol Glacial acetic acid SSC HCl Tween-20 Rabbit anti-FITC antibody DAPI
Staining Vats Wet Boxes Rubbermaid Contrast Microscope Water Bath Oven Micro Pipette Gun Microcentrifuge Centrifuge Tubes Refrigerator RxFISH Cyto Vision System Fluorescence Microscope Cold CCD Camera

Move

I. Specimen preparation and aging

1. Cell suspensions were prepared by methanol/ice acetic acid fixation.

2. Place a drop of the cell suspension on a slide and dry the specimen at the appropriate temperature (25°C) and humidity (45%?50%) required for chromosome spreading.

3. Check the quality of the specimen slides with a phase contrast microscope and draw on the slide areas of good chromosome dispersion or intermediate chromosomes of interest.

4. Age the specimens prepared on the same day by placing them in a 60°C oven for 2 h. Alternatively, age 1d-old specimen slices at room temperature overnight by baking them in a 60°C oven for 1h. Leave the aged specimens at room temperature for subsequent RxFISH steps.

5. Dehydrate specimens in 70%, 85% and 100% series of ethanol for 2 min each at room temperature and air dry.

Preparation of solutions for denaturation and hybridization

1. Prepare 70% ethanol in a 20°C refrigerator.

2. Prepare 50 mL of 70% formamide/2XSSC (pH 7.5) in a 72°C water bath.

3. Prepare 70%, 85% and 100% ethanol and store at room temperature.

4. Prepare wet box and preheat at 37°C.

Probe denaturation

1. Preheat the centrifuge tube containing the RxFISH probe for 5 min at 37°C in a water bath.

2. Mix gently and centrifuge briefly.

3. Pipette with a sterile tip at a probe volume of IOyL per specimen into a 0.5 mL centrifuge tube and cap the tube.

4. Return unused RxFISH probes to 20°C.

5. Denature the RxFISH probe in a water bath at 65°C for lOmin.

6. Place the denatured probe in a 37°C water bath for IOmin?2 h and set aside.

Specimen denaturation

1. Ensure that the temperature of the 70% formamide/2XSSC solution in the staining vat is the desired 72°C (see Notes 1 and 2).

2. Denature specimens (no more than 4) in 70% formamide/2XSSC solution for 1.5 min (see Note 3).

3. Quickly quench denatured specimens in ice-cold 70% ethanol at 20°C (see Note 4).

4. Dehydrate the specimens in 70%, 85% and 100% series of ethanol for 2 min each at room temperature and air dry.

V. Hybridization

1. Reduce the ambient brightness to avoid photobleaching of the RxFISH probe (see Note 5).

2. Take 10uL of the denaturing probe mixture and add it to the hybridization region (containing well-dispersed chromosomes or the mid-dissection phase of interest) drawn on the specimen slice.

3. Cover the 25 mmX25 mm coverslip slowly to avoid air bubbles.

4. Allow the probe solution to spread to the edge of the coverslip and gently snap the coverslip to remove air bubbles without moving the coverslip.

5. seal the slide with rubber cement.

6. Incubate for 48 h at 37°C in a wet box (see Note 6).

VI. Preparation of solutions for post-hybridization steps

1. Prepare 150 mL of 2XSSC solution (pH 7.0) in 3 staining cylinders in a 45°C water bath (see Note 7).

2. Prepare IOOmL of 50% formamide/0.5XSSC solution (pH 7.0) in 2 vats at 45°C in a water bath.

3. Dispense 50 mL of 4:XTween solution (500 mL of 4XSSC and 0.25 mL of Tween-20 stock solution) at pH 7.5 into 1 vat and place in a 45°C water bath.

Elution after hybridization

1. Carefully remove the rubber slime with forceps and incubate the specimen in 2XSSC in the first vat at 45°C for 5 min, sliding off the coverslip (see Notes 8~10).

2. Wash the specimen in 50% formamide/0.5XSSC solution for 5 min, transfer the specimen to the next vat of the same solution and repeat the washing for 5 min.

3. Wash the specimen 51^11 in 2\83(^ solution, transfer the specimen to the next vat of 2\38(: solution for 5 min04. Incubate the specimen at 45°C for IOmin in 4XTween solution.

VIII. Antibody detection steps (optional)

1. At the same time as the second formamide wash in 3.7 above, centrifuge the two antibodies for FITC detection at 14OOOOg for lOmin at room temperature and use only the supernatant for the following experiments.

2. Add 1ul of rabbit anti-FITC antibody and 199uL of 4 X Tween solution to a small centrifuge tube and gently mix the dilutions. Incubate for 10 min at room temperature away from light.

3. Shake off excess liquid from the specimen.

4. Add 200ul of rabbit anti-FITC antibody 1:200 dilution to each specimen slice and incubate for 30 min at 37℃ in a wet box.

5. Reduce the temperature of the water bath from 45°C to 42°C, preheat the 50 mL staining vat with 4XTween solution, and preheat 250 mL of 45XTween solution for 6 elutions.

6. check to make sure that the water bath and 4XTween solution are at 42°C. Then remove the cover slip from the specimen and wash the specimen for 5 min. pour off the solution, add freshly pre-warmed 4XTween solution, and wash the specimen again for 5 min. repeat the procedure once.

7. During the elution process, add 2uL of FITC-labeled goat anti-rabbit IgG antibody and 198juL of 4XTween solution to another centrifuge tube and gently mix the dilution. Incubate for lOmin at room temperature, protected from light.

8. Shake off excess liquid from the specimen.

9. Add 20(^LFITC-labeled goat anti-rabbit IgG antibody 1:100 dilution to each specimen and incubate for 30 min at 37°C in a wet box.

10. Check the temperature of the water bath and 4XTween solution at 42°C and wash the film 3 times for 5 min each time.

IX. DAPI re-staining

Add 10UL of compound and anti-quencher solution to the specimen and seal with a coverslip (22 mmX22 mm) (see Note 11).

X. Image Capture and Analysis

1. Turn on your computer and select the RxFISH software under the Research menu. After opening the software, select the capture interface. Before starting image capture, set the color filter wheel to the appropriate order. The captured probe signals are saved in the original image layer and combined to obtain the final image. the Rx_Cy5 signal is captured first, followed by Rx-Cy3, Rx-FITC and Rx-DAPI.

2. Intermediate scans are performed with FITC. the FITC fluorescence signal is not easily quenched and can be kept bright for all analytical procedures. When an interim is found, focus under a 100X oil lens and select Rx-Cy5.

3. Rx-Cy5 is not visible to the naked eye and relies on computerized images for detailed information. When the exposure is set to 5s, you should be able to detect a faint white image against a dark background. Focus slowly to increase sharpness because there is a time delay between the camera and the software. Adjust the light and dark until you get good contrast (about 90% accuracy). Slight adjustments should be made for different image settings.

4. Click the "Live" button and the filter wheel will automatically move to the next fluorescent filter in the list. In most cases, a 2s exposure for the other fluorescent signal is sufficient. Once you have a clear banded image, click the "Capture" button.

5. After capturing all 4 burst signals, select the screen labeled "RxFISH Image Capture". In the selected screen, complete the synthesis of the whole set of images. Save all the images and do not delete them even if prompted, as they are very useful for analysis.

6. Activate the "Analysis" menu and select the cell you wish to analyze. Selecting "Loadcell" will display chromosomes with multicolored bands (see Note 12). Under the "AnalysisCustomize" menu, the RxFISH image can also be displayed as a DAPI inverted image, which can help to identify chromosomes. The "AnalysisProfile" tool is used to display the intensity profile for each chromosome.

7. Chromosomes can be separated using the "Analysis" command. As soon as the chromosomes are separated, the "Classifier" and "Auto" commands can be used to generate a karyotype of the cell.

Notes

1. The denaturation step is very critical in the operating procedure. The denaturation time and temperature are determined by the type of specimen and the age of the slice. Each experimenter should determine the appropriate temperature and time according to his/her needs. Generally, good results can be obtained within the temperature range of 70-75°C and the time range of 1.5?2 min.

2. Throughout the experiment, the temperature of the liquid in each vat should be checked before use. Usually the temperature of the liquid in the vat is at least TC lower than the temperature of the water bath.

3. Each room temperature specimen placed in 72°C formamide will lower the temperature of the 70% formamide/2 X SSC solution by 0.5-1°C; if more than 4 specimens are placed at a time, the temperature of 70% formamide/2XSSC will not be sufficient for denaturation.

4. If more than 4 specimen slices are processed, the 70% formamide/2XSSC and the first vat of 70% ethanol must be brought back to their initial temperatures, i.e., 72°C and 20°C, respectively.

5. Cy5 fluorescent dye is very sensitive in the infrared wavelength range and is easily excited by white light, therefore it should be captured first during image capture as it will be the first to quench the fluorescence.

6. Hybridize the specimen in a wet box at 37°C for 12~96 h. Do not hybridize the probe for less than 12 h, as this will greatly reduce the signal intensity. Hybridization for 48 h results in a stable, strong signal, which is necessary for successful image capture. The hybridization time can be reduced according to the experience of each laboratory.

7. To ensure the quality of the results, pour off the used solution every day. Solutions used in the denaturation step should not be stored for use in the post-hybridization elution step.

8. The post-hybridization elution step should be done under low light conditions to avoid photobleaching of the directly labeled bulky dyes (Cy5 and Cy3).

9. During all experimental steps after hybridization, the specimen slides should not be dried.

10. If the cover slip does not slide off the carrier sheet, place the specimen sheet in 2XSSC and elute again for 5 min, then check if the cover slip slides off.

11. Store the specimen slides at 1 20°C under light for photographing.

12. If the chromosome color banding is faint and not bright enough, recapture the image at a darker setting (reduce exposure time). Adjust the focus before image capture and shorten the exposure time to minimize delay.

The camera has a chip that collects the fluorescence and passes the information to the computer, which is why there is a delay.The DAPI image should not be too dark; it provides a background for the other fluorescent colors. If the DAPI image is not bright enough, the chromosomes look jagged at the edges. For the same reason, it is necessary to adjust the focus of the DAPI image!

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