Preparation of metaphase slides for molecular cytogenetic experiments

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Preparation of metaphase slides for molecular
cytogenetic experiments
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Criteria of good slides for molecular cytogenetic
experiments

• Longer chromosome
• Well spreading without too much overlapping
  chromosomes
• Little cytoplasmic backgrounds

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Longer chromosomes

• Synchronized culture
• Methotrexate (MTX) blocking method
• Thymidin methods
• EtBr methods

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Chromosome spreading

• Factors influencing the spreading
• Relative humidity
• Temperature
• Drying time of the slides
• Distance of dropping cell onto slide

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Dynamics of chromosome spreading
Time
Fixative layer
a
b
Time
d
c
Spurbeck et al.,1996
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Basel et al.,1997
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• Main factor drying time of fixative solution
  (methanol, acetic acid)
• Relative humidity
• Temperature

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Thermotron CDS-5 cytogenetic drying chamber
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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

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More fixative or acetic acid
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A new method
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Storage of slides

• Short term 4oC in EtOH
• Long term -20oC
• in EtOH or
• in a box filled with N2 gas

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Aging of slides

• Oven aging for G banding
• Room temperature aging
• about two weeks for CGH
• Few days for FISH
• Chemical aging

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Chemical aging

• 2X SSC at 37oC for 3060 min
• EtOH aging

RT?94oC for 2-20s ?RT (1-2oC/s)
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Thank You