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Fluorescence In Situ Hybridization on Metaphase Chromosomes (For biotinylated probes)

Solutions (Pre-made buffers and hybridization kits available for purchase)

  • Hybridization buffer: To 5.5 ml formamide, add 1 g Dextran sulfate and 0.5 ml 20X SSC. Heat to 70oC for several hours to dissolve dextran sulfate, then allow cooling. Adjust pH to 7.0 and bring the volume to 7.0 ml. and store at -20oC. This volume is 70% of that used in the final hybridization mixture; Together with the A.L.Tech probe, this makes the remaining 30% of the volume. Used in this way, hybridization buffer gives a hybridization mixture that is 55% formamide/10% dextran sulfate/1XSSC.

  • Denaturing solution: 70% formamide, 2XSSC, (pH 7). Fresh denaturing solution should be made weekly and stored at 4oC between uses.

  • Ethanol: 70%, 85%, and 100% ethanol in three separate coplin jars.

  • PNM Buffer: 0.1M NaH2PO4 , 0.1M Na2HPO4, 5% Nonfat dry milk, 0.1% NP-40, 0.02% NaAzide. Heat to 37oC for 30 minutes, then leave at room temperature overnight. Centrifuge, save the supernatant, and refrigerate. If more solids form, repeat spin.

  • Washing buffer: 150ml of 50% formamide, 2X SSC (pH7). Divide between three coplin jars. The same jar can be used for the first wash, second wash, and third wash each time. The solutions may be stored at 4oC between uses for several weeks.

  • 4X SSC: 0.05% Tween20

  • Avidin-FITC, Anti-Avidin-FITC. Avidin: Fluorescein Avidin DCS (cell sorter grade) Cat. No. A-2011. Anti-Avidin: Fluorescein Anti-avidin D. Cat. No. SP-2040. Product of Vector Laboratories, Inc.

  • DAPI and anti-fade solution

Preparation of Slides Peripheral blood culture and metaphase spreads are prepared by standard cytogenetic methods. Do not bake the slides. The slides may be treated in 2XSSC for 30 minutes at 37oC or left out at room temperature for a few days to age.

Day 1 Pre-warm denaturing solution at 70oC for 30 minutes. Turn on slide warmer and incubator before beginning the next steps.

  • Probe Denaturation

    1. Add 3ul of A.L.Tech probe and 7ul hybridization buffer in 0.5ml tube to make 10ul hybridization mixture. This 10ul hybridization mixture will be good for one assay under one of 22 mm˛ square coverslip. Caution: Over-diluting our probe may result in weak or no signals.

    2. Mix hybridization mixture well.

    3. Denature the hybridization mix at 70oC for 5 minutes.

    4. Pre-anneal probes by incubating the tubes of hybridization mixture at 37o for 12-15 minutes. (If first FISH results show heavy background noise, pre-anneal for 25-40 min.)

  • Slide Denaturation Meanwhile, denature chromosomal DNA on slides as follows:

    1. Immerse the slides in denaturing solution (70% formamide, 2X SSC, pH 7) for 2 minutes at 70oC.

    2. Pass the slide through the 70%, 85%, and 100% ethanol for 1 minute respectively for dehydration.

    3. Air-Dry the slide and pre-warm the denatured slides on 37oC slide warmer before hybridization.

  • Hybridization

    1. Put the 10 ul hybridization mix onto the slide and apply a 22X22mm˛ coverslip.

    2. Seal the edge of coverslip with rubber cement.

    3. Keep the slide in a moist chamber overnight at 37oC. Day 2 Preheat washing solution at 45oC for 30 minutes. Dilute Fluorescein Avidin (1:200 dilution with PNM buffer) and Fluorescein anti-Avidin (1:100 dilution with PNM buffer).

  • Post-Hybridization Wash

    1. Remove slides from incubator and carefully peel off the rubber cement with forceps. Gently remove coverslip.

    2. Wash the slide with washing buffer (50% formamide, 2XSSC) at 45oC for 3 times, 5 minutes each time.

    3. Wash the slide again with 4X SSC/0.05% Tween20 for 3 times, 2 minutes each time, then follow by washing with 4X SSC for 2 minutes at room temperature.

  • Fluorescence staining Note: perform in the DARK

    1. Place 100ul diluted Fluorescein Avidin (1:200 dilution with PNM buffer) onto slide and apply 24X60mm coverslip or parafilm.

    2. Keep the slide in a moisture chamber in DARK for 20 minutes at room temperature.

    3. Wash the slide again with 4XSSC: 0.05% Tween20 for 3 times, 2 minutes each time, then follow by washing with 4X SSC for 2 minutes at room temperature.

    4. Add 50ul Fluorescein anti-Avidin (1:100 dilution with PNM buffer) and apply coverslip. Keep the slide in a moisture chamber for 20 minutes at room temperature.

    5. Wash the slide again with 4X SSC:0.05% Tween20 for 3 times, 2 minutes each time, then follow by washing with 4X SSC for 2 minutes at room temperature.

    6. Background staining. Put 40ul anti-fade solution (0.5 ug/ml PI or DAPI as counterstain) onto slide and apply coverslip.

    Check the signals under fluorescence microscope or keep in dark box at 4oC.