In the three-color PRINS procedure, three sequential PRINS reactions are performed, each labeling one specific chromosome. The following labeling order is used: (1) FITC for the first targeted chromosome; (2) TRITC for the second targeted chromosome; and (3) FITC for the third targeted chromosome.
1. Prepare a reaction mixture in a final volume of 50 pL containing: 0.2 mMdATP, dCTP, and dGTP, 0.02 mMdTTP, 0.02 mMFITC-12-dUTP, 50 mMKCl, 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl2, 0.01% BSA, 200 pmol of oligonucleotide primer; and 2.5 U of Taq DNA polymerase. In practice, mix in a sterile microcentrifuge tube: 1 pL of each 1:10 diluted dATP, dCTP and dGTP; 1 pL of the 1:100 diluted dTTP; 1 pL of FITC-12- dUTP, 1 pL of BSA; 5 pL of 10X Taq buffer; 0.5 pL of the Taq DNA polymerase; 4 pL of the specific primer (for instance, the specific primer for chromosome 1 according to the procedure illustrated in Fig. 1, Chapter 6), and distilled water to 50 pL.
2. Place the reaction mixture under a 22 x 32 cover slip on the denatured slide, and transfer to the heating block of the thermal cycler.
3. Set up the PRINS program and start the reaction. The program consists of a unique 5 min step at the specific annealing temperature of the primer involved for both in situ annealing and elongation.
4. While this first reaction is running, prepare the reaction mixture for the second PRINS reaction as described above, but incorporating the specific primer for the second targeted chromosome, and TRITC-6-dUTP.
5. On completion of the program, carefully remove the cover slip from the slide.
6. Wash the slide twice for 2 min at room temperature in 1X PBS.
7. After draining the excess 1X PBS off the slide, and before the slide is completely dry, put the second PRINS reaction mixture on the slide, and cover with a 22 x 32 cover slip.
8. Place the side again on the plate of the thermal cycler.
9. Set up the program for the second PRINS reaction: 5 min at the annealing temperature, specific to the second primer used.
10. Start the program.
No additional denaturation is required after the first PRINS reaction because the chromosomal DNA remains denatured through the PRINS incubations.
11. While this second reaction is running, prepare the reaction mixture for the third PRINS reaction, incorporating the specific primer for the third targeted chromosome and FITC-12-dUTP.
12. At the end of the second reaction, remove the cover slip from the slide and repeat the washing as in steps 6 and 7.
13. Before the slide is completely dry, put the third PRINS reaction mixture on the slide, and cover with a 22 x 32 cover slip.
14. Place the slide on the thermal cycler.
15. Set up the program for the third PRINS reaction: 5 min at the annealing temperature, specific to the third primer used.
17. At the end of this third reaction, the slide is transferred to 4X SSC, 0.05% Tween-20 for two washes (3 min each) at room temperature with gentle agitation.
18. Drain the excess washing solution off the slide.
19. Mount the slide in Vectashield antifade solution containing either DAPI (0.3 |L/mL) or a mix of propidium iodide (0.3 |L/mL) and DAPI (0.3 |L/mL).
20. Cover with a 22 x 40 cover slip and seal the cover slip with rubber cement.
21. Examine the slide under the epifluorescence microscope, preferentially using first the triple band-pass filter, and confirming the coloration of the fluorescent spot with single band-pass filters.
22. The slide may be stored in the dark at 4°C for several months.
1. Although some protocols propose the use of ethanol instead of methanol, because of the extreme toxicity of this compound, the fixative mixture prepared using methanol is optimal for tissue preparation (especially brain tissues) for molecular cytogenetic analysis.
2. Instead of Tween-20, another mild detergent, such as Triton-X100, may be used (0.1% of Triton-X 100 should be added in SSC solution).
3. This step can be skipped when postmortem brain samples are processed.
4. It is recommended that one leave approx 2 mL of liquid above the pseudo-solid phase.
5. The number of repetitions of step 6 should be assessed empirically and principally depends on the age of sample (e.g., freshly prepared autopsy requires to repeat fixation three times; however, for autopsies older than 48 h, repeating the fixation step five or six times is recommended).
6. The conditions of pepsin treatment generally are selected empirically; however, it should be noted that some of formalin-fixed and paraffin-embedded sections of brain could require a long-term pepsin treatment (as long as 7 min).
7. The protocol proposed also can be applied to frozen sections of brain tissues. For frozen sections, skip steps 1-4.
8. Tissue section should be 7 to 14 |M to provide a sufficient amount of nuclei to analyze and reduce the problem of damaged nuclei because the size of brain tissue cells is quite large compared with a number of another somatic tissue cells.
9. The leading cause of problems in preparation of formalin-fixed autopsic brain tissue is the incomplete DNA-peptides complex disruption during processing of the slides for molecular-cytogenetic analysis. The treatment with NaSCN solution should be conducted and conditions of the step are selected empirically.
10. RNase treatment is especially recommended for frozen sections of brain tissue. The step can be skipped for frozen sections kept in liquid nitrogen or fresh paraffinized sections as well as when DNA probes for nontranscribed DNA sequences are applied (6 ).
11. Quality control procedure can be applied to cell suspensions only.
12. Sudan black solution is used for postmortem brain samples only. Postmortem brain suspensions usually are characterized by increased level of lipofuscin-like autofluorescence. Treatment with Sudan black solution reduces considerably the lipofuscin-like autofluorescence and does not affect specific fluorescence labels (7).
We thank Viktor V. Monakhov and Alexei D. Kolotii for technical assistance. The work is supported by INTAS 03-51-4060.
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