Fluorescence in situ hybridization using single-copy genomic probes and chromosome coloring methods
Fluorescence in situ hybridization using single-copy genomic probes and chromosome coloring methods
Hybridization of biotin-labeled or digoxigenin-labeled probes to chromosomes detects degenerate chromosomes, and individual probes can be detected by double-antifluorescence staining.
Operation method
Scheme 27.9 Fluorescence in situ hybridization (FISH) using single-copy genomic probes and chromosome painting methods
Materials and Instruments
SSC D-PBSA Glycogen RNase Paraformaldehyde Formamide Move Precipitation of probes containing repetitive sequences: Caveat The correct combination of antibodies must be used, e.g. if a digoxin-resistant sheep polyantiserum is used as the primary antibody, the secondary antibody must be a FITC-coupled donkey anti-sheep IgG antibody. For more product details, please visit Aladdin Scientific website.
Fixative Ethanol Hybridization Buffer Labeling Probes Rubber Emulsion Adhesive Sealer
Large molecular weight mucoid probes often contain repetitive sequences, and failure to sequester these sequences prior to hybridization will result in a severely non-specific hybridization background. The addition of human Cot1 DNA, which is rich in repetitive sequences, eliminates this effect by competitively inhibiting the repetitive sequences of the mucilage. Cot1 DNA can be precipitated in step (b ) below.
(a) For a 20 μl nick-pan labeling reaction system, take 1 μl of 0.5 mol/L EDTA, 2.5 μl of 4 mol/L LiCl, 1 μl of glycogen, 100~1000-fold excess of human Cot1 DNA (Invitrogen), and 100 μl of ethanol, and mix them with labeled probes.
(b) Place on dry ice for 30 min or -20 ℃ overnight to precipitate.
(c) Centrifuge for 15 min to precipitate the DNA.
(d) Wash the precipitate with 70 % ethanol.
(e) Reprecipitate the DNA by centrifugation and dry the DNA at room temperature.
(f) Redilute the DNA in hybridization buffer at a concentration of 2-10 ng/μl.
Precipitation without repeat probes:
Same method as previously described for Kimi, but Cot1 DNA needs to be removed from the precipitate.
Probe denaturation:
(a) For probes containing repetitive sequences, they need to be blocked with Cot1 DNA. Heat the probe mix to 70°C for 10 min. Allow the probe mix to stand at 37°C for 1 h before applying to slides.
(b) For probes that do not contain repeats, heat the Probe Mix to 70°C for 10 min and then cool it on ice for 10 min.
TBST buffer for detection of probes, containing 0.05% Tween20 (Tween), prepared with 0.1 mol/ L Tris, 0.15 mol/L NaCl (pH 7.5)
formamide, and formulated as 50%
antibody with 1X SSC:
(a) Primary antibody (monoclonal antibody), e.g., anti-digoxin or anti-biotin sheep polyantibody serum; see product description for titration (Roche)
(b) Secondary antibody (secondary antibody), e.g. FITC-coupled donkey anti-sheep IgG (JacksonlmmunoresearchInc.).
(c) Dilution of antibody with TBST containing 3% bovine serum albumin (BSA )
Chromosome preparation and denaturation:
1. Prepare medium-term quiescent cells using standardized techniques (see Scheme 16. 7 and Scheme 27. 5) by dropping the fixed cells onto a slide and marking the area with a diamond etching pen.
2. Re-fix the cells with freshly prepared methanol and acetic acid (3 : 1 ) fixative for 1 h, then air-dry the slides.
3. Wash the slide with 2X SSC for 2 min at room temperature.
4. incubate the slides with 2X SSC containing 100 μg/ml of RNase for 1 h at 37°C.
5. Rinse with D-PBSA.
6. Re-fix with freshly prepared D-PBSA containing 1 % paraformaldehyde for 10 min at room temperature (this procedure is optional).
7. Rinse with D-PBSA.
8. Perform two rounds of dehydration with 70% and 100% ethanol for 2 min each and air dry the slides.
9. Denature the chromosomes with 2X SSC containing 70% formamide for 2-4 min at 70°C (try from 2 min to determine the appropriate time for the experiment), making sure that the temperature is 70°C before using 70% formamide.
10. Wash the slides in batches with ice-cold 70% ethanol.
11. Repeat the dehydration procedure in step 8 and air dry the slide.
12. The chromosomes are now ready for hybridization.
Hybridization:
13. Add 10 μl of denaturing probe (labeling probe: large molecular weight mucoid clones are well suited as probes for single-copy genes, but cDNA probes can also be used. The DNA is labeled by notch panning using a commercial kit; Roche (formerly BoehringerMamiheim) produces a notch panning kit for biotin or digoxin doping, see product description for details). For details, refer to the product description), cover this area with a 22 mm X 22 mm coverslip.
14. Close the coverslip around the perimeter with rubber latex adhesive.
15. Place the slide in an incubator at 37°C saturated humidity overnight.
Probe Assay:
16. On the following day, the slides were removed, immersed in 2X SSC, the coverslips were removed (at room temperature), the adhesive around the periphery was removed, and the slides were placed in a Coplin bottle.
17. Soak the slides in 1X SSC containing 50 % formamide for 10 min twice at 42°C.
18. The slides are washed twice with 2X SSC for 10 min each time at 42°C.
19. Rinse with TBST.
20. Incubate the slides in TBST containing 3% bovine serum albumin for 30-60 min at 37°C to block non-specific binding.
21. 100 μl of antibody per slide was added dropwise to the slides, and each slide was covered with Parafilm.
22. Incubate slides for 1 h at 37°C.
23. Wash the slides with 500 ml of TEST at room temperature.
24. Add 3% BSA/TBST diluted fluorescein-coupled secondary antibody to the slide, see product instructions for potency (titer) of the secondary antibody or feel free to use it yourself.
25. Incubate at 37°C for 30 min.
26. Wash with 500 ml TBST for 30 min at room temperature.
27. Re-stain with TBST containing 0.8 μg/ml diaminophenylindole (DAPI) and/or 0.4 μg/ml propylene iodide (PI) for 10 min and cover with an anti-discoloration slide.
28. Cells on slides were visualized by fluorescence microscopy through the appropriate filter combinations.