Fluorescence in situ hybridization (FISH) on metaphase chromosomes

1. preparation of metaphases

  • incubate a large flask of TOU lymphoblast cells until the cells are fast growing
  • split the flask and add fresh medium, incubate over night
  • add 300 µl colcemid (10 µg/ml) to 30 ml culture medium and incubate the cells 2...2.5 hours at 37°C under CO2
  • centrifuge 10 min 1000 rpm, remove the supernatant
  • add 40 ml 0.4 % KCl and incubate 30 min at 37°C
  • centrifuge 10 min 1000 rpm, remove the supernatant
  • wash 4x in 40 ml freshly prepared fixative (methanol/glacial acetic acid = 3/1)
  • store in 10 ml fixative at -20°C

2. preparation of metaphase spreads

  • wash the cells with 10 ml freshly prepared fixative (methanol/glacial acetic acid = 3/1), do not exceed 1000 rpm for the centrifugation
  • resuspend the cells in 1...2 ml fixative
  • clean the slides with fixative and dry them
  • drop 20 µl cell suspension on the dry slide
  • (just after evaporation of the fixative dip the slide in 70 % acetic acid or fixative, incline the slide and let the acid run off. This should remove most of the cytoplasm and cell debris but can also detach the chromosomes.)
  • dehydrate the sample in 50 %, 70 % and 96 % ethanol (2...5 min each)
  • air dry
  • check the slides for quality and density of metaphases

3. pretreatment of metaphase spreads

  • drop 20 µl RNAse solution (1 ml: 100 µl 20xSSC, 10 µl 10 g/l RNAse, 890 µl ABD) on the slide, cover with a cover slip or parafilm and incubate 1 hour in a moist chamber at 37°C
    (The existence of a high RNA background can be tested by staining with propidium iodide. PI with stain both RNA and DNA, DAPI stains only DNA.)
  • wash the slide 5 min in 2xSSC
  • dehydrate the sample in 50 %, 70 % and 96 % ethanol (2...5 min each)
  • air dry
  • prepare a joplin jar with pepsin solution (100 ml: 4 ml 0.25 N HCl, 50 µl 10 % pepsin, 96 ml ABD)
  • incubate the slides 30 min at 37°C
  • wash the slides 5 min in postfixation wash-buffer (100 ml: 10 ml 10xPBS, 2.5 ml 2 M MgCl2, 87.5 ml ABD) at RT
  • incubate the slides 5 min in postfixation buffer (100 ml: 10 ml 10xPBS, 2.5 ml 2 M MgCl2, 10.8 ml 37 % Formaldehyde, 76.7 ml ABD) at RT
  • wash the slides 5 min in PBS
  • dehydrate the sample in 50 %, 70 % and 96 % ethanol (2...5 min each)
  • air dry
  • check the slides for quality and density of metaphases

4. hybridization

satellite and unique sequences

unique + repetitive sequences
  • nick translation of 2 µg probe in 50 µl (e.g. Gibco Kit)
  • nick translation of 2 µg probe in 50 µl
  • stop reaction with 5 µl 0.5 M EDTA
  • precipitation with 2 µl salmon sperm DNA (20 g/l), 5.5 µl 3 M NaOAc, 150 µl EtOH
  • -20°C 30 min
  • centrifugation, wash with 70 % EtOH, air dry
  • resolve in 100 µl hybridization buffer (50 % formamide, 2x SSC, 10 % DS)
  • stop reaction with 5 µl 0.5 M EDTA
  • precipitation with 100 µl Cot1 DNA (1 g/l), 2 µl salmon sperm DNA (20 g/l), 15 µl 3 M NaOAc, 420 µl EtOH
  • -20°C 30 min
  • centrifugation, wash with 70 % EtOH, air dry
  • resolve in 100 µl hybridization buffer (50 % formamide, 2x SSC, 10 % DS)

  • 5 min denaturation in PCR machine at 80°C
  • 5 min on ice
  • 30 min pre-annealing at 37°C
  • apply a total of 10 µl of probe solution in hybridization buffer (satellite 2 µl, unique 10 µl probe) on slide and cover with cover slip
  • seal the cover slip with rubber cement
  • 5 min denaturation at 75°C (in-situ PCR chamber)
  • 5 min denaturation of metaphase DNA in 15 µl denaturation puffer (1 ml: 700 µl ultra-pure formamide, 100 µl 20xSSC, 200 µl ABD) at 80°C (in-situ PCR chamber) covered with cover slip
  • 5 min in ice-cold 70% EtOH
  • dehydration in 96% EtOH
  • air dry on hot plate (37°C)
  • apply the probe, cover and seal the cover slip with rubber cement
  • incubation over night in moist chamber at 37°C
  • incubation over night in moist chamber at 37°C
5. Detection

  • 15 min wash the slides in 200 ml 50% formamide/2xSSC (42°C for alpha-sat, 37°C other. Temperature inside the jar!)
  • 15 min wash in 200 ml 2xSSC at 37°C
  • for alpha-sat: wash in 0.1xSSC at RT
  • 15 min wash in 200 ml 4xSSC/0.03 % Tween 20 at 37°C
  • apply 10 µl blocking solution (5 % BSA in 4xSSC 0.03 % Tween 20), cover with cover slip and incubate at 37°C for 30 min
  • (centrifuge antibody solution 3 min)
  • apply 10 µl antibody solution (recommended dilution in 5 % BSA in 4xSSC 0.03 % Tween 20), cover with cover slip and incubate at 37°C for 30 min
  • 15 min wash in 200 ml 4xSSC/0.03 % Tween 20 at 37°C
  • (centrifuge secondary antibody solution 3 min)
  • apply 10 µl secondary antibody solution, cover with cover slip and incubate at 37°C for 30 min
  • 15 min wash in 200 ml 4xSSC/0.03 % Tween 20 at 37°C
  • (centrifuge antibody solution 3 min)
  • apply 10 µl antibody solution, cover with cover slip and incubate at 37°C for 30 min
  • 15 min wash in 200 ml 4xSSC/0.03 % Tween 20 at 37°C
  • dehydrate the sample in 50 %, 70 % and 96 % ethanol (2...5 min each)
  • air dry
  • embedding in 10 µl VECTASHIELD with DAPI or/and PI

Go back