Preparation of Interphase Nuclei Suspension From the Specimens of Fetal and Adult Brain Tissues

1. Taped homogenizer: Teflon pestle and glass tube (Cole-Parmer International; cat. nos. A-04368-32 for Teflon pestle and A-04368-33 for glass tube).

2. Earle's buffered saline solution (EBBS; cat. no. 24010-035, Gibco Invitrogene, SARL; Cergy Pontois Cedex, France).

3. Solution of phosphate-buffered saline (PBS) pH 7.3, containing 0.1% (w/v) of Nonidet P-40. PBS preparation: prepare 10X stock water solution with 1.37 M NaCl, 27 mM KCl, 100 mM Na2HPO4, and 18 mM KH2PO4 (pH 7.4 is adjusted by 1 N HCl). The solution is stored at room temperature.

4. 60% (w/v) solution of glacial acetic acid.

5. Fixative mixture of methanol:glacial acetic acid (3:1, v:v) freshly prepared and stored at -20°C. Caution: methanol is extremely toxic (see Note 1).

6. Ethanol 100% and 96%, and water-diluted ethanol to 70%.

7. Pepsin solution: 10% (w/v) pepsin solution (stored at -20°C) is diluted in prewarmed (37°C) solution of 0.01 NHCl (chlorohydric acid). Pepsin solution is made fresh as required.

9. Microscope slides, 25 x 75 x 1 mm, plain.

2.2. Preparation of Cell Suspension From Cultured Fetal Neuronal Cells (Short-Term or Long-Term Fetal Brain Cultures and Neuronal Stem Cell Cultures)

1. Hypotonic solution (0.9% NaCl water dissolved).

2. EBBS (cat. no. 24010-035, Gibco Invitrogene).

3. Fixative mixture of methanol:glacial acetic acid (3:1, v:v) freshly prepared and stored at -20°C. Caution: methanol is extremely toxic.

4. Ethanol 100%, 96%, and water-diluted ethanol to 70%.

5. Pepsin solution: 10% (w/v) pepsin solution (stored at -20°C) is diluted in prewarmed (37°C) solution of 0.01 NHCl (chlorohydric acid). Pepsin solution is made fresh as required.

7. Microscope slides, 25 x 75 x 1 mm, plain.

2.3. The Preparation of Formalin-Fixed and Paraffin-Embedded Sections of Brain

1. Ethanol 100%, 96%, and water-diluted ethanol to 70%.

2. 20X Standard saline citrate (SSC): 3 M sodium chloride, 0.3 Mtrisodium citrate. The solution is stored at room temperature. The working solution is prepared by dissolving one volume of 20X SSC in four volumes of water and adding Tween-20 to 0.5% (see Note 2).

3. Pepsin solution: 10% (w/v) pepsin solution (stored at -20°C) is diluted in prewarmed (37°C) solution of 0.01 N HCl. Pepsin solution is made fresh as required.

4. PBS solution.

5. PBS/MgCl2 working solution: 1 volume of 2 MMgCl2 in 38 volumes of 1X PBS.

6. Formaldehyde/PBS/MgCl2 solution: Add 2.7 mL of formaldehyde (37%) in 100 mL of PBS/MgCl2 solution (to produce 1% of formaldehyde in PBS/MgCl2 working solution).

8. Sodium isothiocyanate (NaSCN) 1 M (w/v) for disruption of DNA-protein complexes. Caution: NaSCN is toxic; the wearing of gloves is indispensable.

9. Deparaffinization solvent: Xylene (100%).

11. Microscope slides, 25 x 75 x 1 mm, plain.

2.4. Quality Control

1. Microscope slides, 25 x 75 x 1 mm, plain.

2. 24- x 24-mm Cover slips (CML France, Nemours Cedex, France).

4. Nuclear stain: 300 nMDAPI in water.

5. Sudan black ethanol-water solution. Dissolve 0.7 g of Sudan black in 100 mL of 96% ethanol, then add and stir with 50 mL of water. The solution is stored at room temperature.

2.5. Multicolor PRINS

1. dATP: 100 mM solution (Roche Diagnostics, Meylan, France) diluted 1:10 with sterile distilled H2O.

2. dCTP: 100 mM solution (Roche Diagnostics) diluted 1:10 with sterile distilled H2O.

3. dGTP: 100 mM solution (Roche Diagnostics) diluted 1:10 with sterile distilled H2O.

4. dTTP: 100 mM solution (Roche Diagnostics) diluted 1:100 with sterile distilled H2O.

5. Fluorescein isothiocyanate 2'-deoxyuridine 5'-triphosphate (FITC-12-dUTP): 1 mM (Roche Diagnostics).

6. Tetramethylrhodamine isothiocyanate (TRITC-6-dUTP): 1 mM (Roche Diagnostics).

8. Bovine serum albumin (BSA; Sigma, St. Louis, MO).

9. Taq DNA polymerase (Roche Diagnostics) or Ampli Taq (Perkin Elmer, Foster City, CA).

10. 10X Taq buffer: 500 mMKCl, 100 mMTris-HCl, pH 8.3, 15 mMMgCl2.

11. Oligonucleotide primers at 50 pmol/pL.

13. Sterile distilled water.

14. Tween-20 (Roche Diagnostics).

15. Washing buffer (diluted from 20X SSC): 4X SSC, 0.05% Tween-20.

16. 1.5-mL Sterile microcentrifuge tubes (Eppendorf AG, Hamburg, Germany).

19. Programmable thermal cycler equipped with a flat pate block (Hybaid Ltd., Teddington, UK).

2.6. Detection and Microscopy

2. Propidium iodide (Sigma).

3. Antifade solution Vectashield (Vector Labs, Burlingame, CA).

5. Rubber cement.

6. Epifluorescence Microscope Zeiss Axioskop (Carl Zeiss) equipped with x40 and x100 Plan Fluo objectives, and with a set of single band-pass filters for DAPI,

FITC, and TRITC single band-pass filter and a triple filter (filter B/G/R, cat. no. 513836) for the simultaneous observation of DAPI, FITC and TRITC signals.

7. Images are captured by a Vysis QUIPS Imaging Analysis System (Vysis, Downers Grove, IL).

3. Methods

3.1. Preparation of Interphase Nuclei Suspension From the Specimens of Fetal and Adult Brain Tissues

1. Place the brain tissue in Petri dish and rinse it in 2 mL of EBBS (see Note 3).

2. Take a piece of the brain tissue (approx size 3 x 3 x 3 mm) and place it into the homogenizer glass tube. Use the Teflon pestle to homogenize the piece by intense rotating of pestle to produce the liquid-like substance.

3. Add into glass tube containing homogenized tissue 2 mL of PBS containing 0.1% (w/v) of Nonidet P-40 and homogenize for 30 s.

4. Put the substance obtained into a 15-mL plastic (or glass) tube and add 1 mL of 60% (w/v) glacial acetic acid. Leave the mixture obtained for 3 to 5 min at room temperature.

5. Add 9 mL of fixative mixture and centrifuge at 1000^for 5 min.

6. Decant supernatant (see Note 4) and add fixative mixture to 10 mL of total solution volume. Centrifuge at 3000^ for 8 min.

7. Repeat step 6 three times (see Note 5).

8. The obtained suspension can be stored in a 2-mL tube for 6 to 12 mo at -20°C.

9. Put 100 pL of suspension obtained on microscope slide and air-dry for 15 to 20 min.

10. Place slides in dilute pepsin solution (20-100 pL of pepsin) for 3 to 5 min (see Note 6).

11. Place slides in PBS for 5 min.

12. Dehydrate through series of ethanol (100%, 96%, and 70%, 3 min each) and air-dry. Then proceed to PRINS procedure.

3.2. Preparation of Cell Suspension From Cultured Fetal Neuronal Cells

1. Take 1 to 2 mL of neuronal cells culture (1-2 millions cells per mL) and centrifuge at 3500^ for 7 min.

2. Wash the precipitation in 1 mL of EBBS by adding it to the tube with precipitant and mixing by inversion for 15 to 30 s. Repeat this procedure using 1 to 1.5 mL of hypotonic solution instead EBBS. Centrifuge at 3500^ for 7 min.

3. For the decant supernatant, add 1.9 mL of fixative mixture and leave the mixture for 20 min at -4°C.

4. Repeat step 3 three times. The resulted suspensions should be kept at -20°C.

5. Put 100 pL of suspension obtained on microscope slide and air-dry for 15 to 20 min.

6. Place slides in dilute pepsin solution (20-100 pL of pepsin) for 3 to 5 min (see Note 6).

7. Place slides in PBS for 5 min.

8. Dehydrate through series of ethanol (100%, 96%, and 70%, 3 min each) and air-dry. Then proceed to chromosomal PRINS labeling procedure.

3.3. Preparation of Formalin-Fixed and Paraffin-Embedded Sections of Autopsy Brain Tissues (see Note 7)

1. The brain tissue section size is supposed to be 7 to 20 pM (see Note 8). Place the slides with mounted brain tissue sections in 100% xylene at room temperature for 5 min, change xylene and repeat for another 5 min.

2. Rehydrate through series of ethanol (100%, 96%, and 70%, 2 min each) and wash in SSC/detergent mixture for 20 min mixed by inversion periodically (the procedures are made at room temperature).

3. Place the slides into Coplin jar containing 1 MNaSCN solution for 3 to 5 h or overnight (see Note 9).

4. Wash slides by water for a few seconds (without drying).

5. Apply 100 pL of RNase solution in 2X SSC to slide under cover slip and incubate at 37°C for 15 to 30 min (see Note 10).

6. Place slides in dilute pepsin solution (20-100 pL of pepsin) for 3 to 5 min (see Note 6).

7. Place slides in 2X PBS, PBS/MgCl2, formaldehyde/PBS/MgCl2, and again in 1X PBS, subsequently each for 5 min.

8. Dehydrate through series of ethanol (100%, 96%, and 70%, 3 min each) and air-dry. Then, immediately proceed to PRINS procedure.

3.4. Quality Control (see Note 11)

1. Drop 5 to 15 pL of prepared suspension on the microscope slide and air-dry.

2. Look into light microscope by using phase contrast option. If the distribution of nuclei is satisfactory (Fig. 1.1, 1.5, 1.6, 1.10, 1.12), skip next two steps.

3. If the distribution of nuclei is too low to analyze (Fig. 1.2, 1.3, 1.8, 1.9), centrifuge at 3500g for 7 min and decrease the volume in the tube twice. Then mix by inversion and repeat step 2. If the distribution of nuclei is satisfactory, skip the next step.

4. If the distribution of nuclei is too dense to analyze (Fig. 1.4, 1.7, 1.11), centrifuge at 3500g for 7 min and decrease the volume in the tube twice. Then, add 0.3 to 0.7 mL of fixative mixture and repeat step 2.

Fig. 1. (oppositepage) Analysis of nuclei density in suspension prepared from adult and fetal brain tissue by phase-contrast microscopy. Suspension of adult brain tissue nuclei, 1.1 to 1.3. (1) Dense distribution of nuclei with a high level of superposition. (2) Twice-diluted suspension with a optimal nuclei density. (3) Suspension diluted three times with a lower nuclei density, 1.4 to 1.6. Suspension of adult brain tissue nuclei. (4) Too-dense distribution of nuclei for molecular cytogenetic analysis. (5,6) Three times diluted suspension with optimal nuclei density. Suspension of adult brain tissue nuclei, 1.7 to 1.8. (7) Extremely dense distribution of nuclei. (8) Four times

Fig. 1. (continued from opposite page) diluted suspension. It should be noted that suspension with low-density nuclei distribution are more suitable for analysis than suspensions with an extremely density of nuclei distribution. Suspension of fetal brain tissue nuclei, 1.9 to 1.10. (9) Low-density nuclei distribution. (10) Concentrated fetal brain nuclei suspension. Suspension of fetal brain tissue nuclei, 1.11 to 1.12. (11) Extremely dense distribution of nuclei. (12) Suspension diluted four times.

Fig. 1. (continued from opposite page) diluted suspension. It should be noted that suspension with low-density nuclei distribution are more suitable for analysis than suspensions with an extremely density of nuclei distribution. Suspension of fetal brain tissue nuclei, 1.9 to 1.10. (9) Low-density nuclei distribution. (10) Concentrated fetal brain nuclei suspension. Suspension of fetal brain tissue nuclei, 1.11 to 1.12. (11) Extremely dense distribution of nuclei. (12) Suspension diluted four times.

5. Drop 10 pL of DAPI water solution on microscope slide with prepared suspension of calibrated nuclei density. Put a 24- x 24-mm cover slip on the drop. Examine at the result in a fluorescent microscope.

6. Put the slide into Sudan black solution for 3 min and then rinse in PBS for 3 to 5 min and repeat step 5 (for postmortem brain suspensions only; see Note 12).

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