1. Preparation of metaphase slides for molecular cytogenetic experiments

2. Criteria of good slides for molecular cytogenetic experiments Longer chromosome Well spreading : without too much overlapping chromosomes Little cytoplasmic backgrounds

3. Longer chromosomes Synchronized culture –Methotrexate (MTX) blocking method –Thymidin methods –EtBr methods

4. Chromosome spreading Factors influencing the spreading –Relative humidity –Temperature –Drying time of the slides –Distance of dropping cell onto slide

5. Dynamics of chromosome spreading Time ab c d Fixative layer Spurbeck et al.,1996

6. Basel et al.,1997

7. Main factor –drying time of fixative solution (methanol, acetic acid) –Relative humidity –Temperature

8. Thermotron CDS-5 cytogenetic drying chamber

9. How we do? Prepare slide in a good weather Slides should be very clean Using dry slide Before or after dropping cell, add one or two drop fixative solution on slides

10. More fixative or acetic acid

11. A new method

12. Storage of slides Short term : 4 o C in EtOH Long term : -20 o C –in EtOH or –in a box filled with N 2 gas

13. Aging of slides Oven aging for G banding Room temperature aging –about two weeks for CGH –Few days for FISH Chemical aging

14. 2X SSC at 37 o C for 30~60 min EtOH aging RT  94 o C for 2-20s  RT (1-2 o C/s)

15. Thank You