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Box 1. Slide pretreatment with pepsin to remove cytoplasm

From the following article

Spectral karyotyping analysis of human and mouse chromosomes

Hesed M Padilla-Nash, Linda Barenboim-Stapleton, Michael J Difilippantonio and Thomas Ried

Nature Protocols 1, 3129 - 3142 (2007)

doi:10.1038/nprot.2006.358

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  1. Equilibrate the slide in a coplin jar containing 2times SSC for 5 min, without shaking, at room temperature. The solution should cover all the slides.
    filled triangle CRITICAL STEP Size of coplin jar used depends on number of slides being pretreated.
  2. Dilute the 20 mg ml-1 RNase A stock solution (slide pretreatment solution 1) 1:200 in 2times SSC, made and kept at room temperature until use.
  3. Apply 120 mul to a 24 mm times 60 mm coverglass, invert slide metaphase-spread-side down onto the liquid bearing coverglass, and reinvert slide, taking care not to scratch the slide.
    filled triangle CRITICAL STEP The purpose of this step is to digest DNA molecules surrounding the chromosomes that would interfere with the hybridization and result in high fluorescent background. Ensure that sample is in full contact with the liquid and that air bubbles have been eliminated.
  4. Incubate slides in a moist, lightproof, waterproof hybridization box (as described in Step 29) at 37 °C for 45 min.
  5. Carefully remove coverglass by gently tapping the sides of slide onto a solid surface; the coverglass should slide off easily.
  6. Rinse the slide by placing in a coplin jar containing 2times SSC for 5 min at room temperature, with shaking.
  7. Repeat Box Step 6 two more times, with shaking (at room temperature), using fresh 2times SSC each time.
  8. Add 2–30 mul pepsin stock solution (slide pretreatment solution 2) into an empty, clean 100-ml glass beaker, then add 100 ml 0.01 M HCl that has been prewarmed to 37 °C in a water bath, and adjust the pH to 2.0 using 1.0 N HCl. Mix well and pour appropriate volume into a coplin jar sufficient to cover slide.
    filled triangle CRITICAL STEP It is very important that the pepsin be added to the clean beaker first and not directly into the HCL. If the pepsin is added to the HCL, it does not dissolve into solution.
  9. Incubate slide in coplin jar for 30 s–5 min at 37 °C.
    filled triangle CRITICAL STEP The time of pepsin treatment and amount of pepsin stock solution to be used is dependent on (i) the amount of cytoplasm surrounding the metaphase spreads, as observed with a light microscope using phase objectives before slide pretreatment, and (ii) the age of the slide—slides with excess cytoplasm, or older than 6 months, may require longer treatment with pepsin (3–5 min) and higher concentrations of pepsin (10–30 mul). After exposure to pepsin, the slide can be placed into a Petri dish containing 1times PBS and examined using an inverted microscope, to see whether longer pepsin treatment is required. If so, place the slide back into the coplin jar containing the pepsin-acid mixture.
  10. Wash the slide in 1times PBS for 5 min at room temperature, with shaking.
  11. Repeat Box Step 10, using fresh 1times PBS.
  12. Wash the slide once for 5 min at room temperature in 1times PBS/MgCl2 (slide pretreatment solution 3), with shaking.
  13. Place the slide in a coplin jar containing 1% (vol/vol) formaldehyde/1times PBS/MgCl2 (slide pretreatment solution 4) and incubate slide (not shaking) for 10 min at room temperature (when finished with procedure decant solution into an appropriate waste container).
  14. Wash slide for 5 min in 1times PBS at room temperature, shaking.
  15. Dehydrate slide in an alcohol series (70%, 90% and 100% ethanol), using different coplin jars, at room temperature, without shaking, for 3 min each.
  16. Air-dry the slide until the ethanol has evaporated (1–2 min), on the workbench.
  17. Check chromosomes to see whether the morphology has been preserved, and select an area for hybridization.
    filled triangle CRITICAL STEP After slide pretreatment, the chromosomes should retain their original morphology and should not have the appearance of being overdigested by the enzyme. If they look hollow, they are not generally useful for SKY hybridization, and there is no reversal of this step. If there are enough slides available, using additional slides with different pepsin concentrations and times is highly recommended to achieve the best results. Redo the entire procedure if the pretreatment does not sufficiently remove the cytoplasm surrounding the chromosome.
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