Preparing slides for FISH

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Preparing slides for FISH

Last updated on
  1. Take about 100 ul blood from mouse into heparanized tube
  2. Dilute blood in 5 ml PBS in a 15 ml conical.
  3. Spin tubes for 7 min at 1000 rpm
  4. Remove supernatant
  5. Gently resuspend cells in remaining volume by flicking tube with finger
  6. Slowly and gently resuspend the cells in 5 mls of 0.075 M KCl, 37oC.
  7. Incubate 5 minutes at 37oC to allow cells to swell
  8. Centrifuge 1000 rpm x 7 minutes
  9. Remove KCl leaving a few drops of the solution and resuspend by tapping with finger
  10. Dropwise with constant mixing, add 5 mls cold freshly prepared fixative (3:1 Methanol:Glacial Acetic Acid)
  11. Vortex for 30 seconds at lowest speed setting
  12. Incubate on ice for 30 minutes
  13. Centrifuge and remove supernatant
  14. Add 5 mls fresh ice-cold fixative
  15. Perform steps 13 and 14 two more times (3 washes total after 30 min incubation)
  16. The final volume of fixative will depend on the cell pellet, and spreads on the slide will need to be checked for cell density
  17. Drop 10-20 ul of cell suspension on a clean microscope slide (high quality precleaned slides, soak in 100% EtOH overnight, wash with distilled H2O and wipe clean with lint free paper towel), drop from height of about 6 inches
  18. Dehydrate the slides through an EtOH series (70%, 90%, and 100% - 5 minutes each), and air dry.
  19. Store slides in box covered with parafilm at -20C
Y-chromosome
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Diane Krause, MD, PhD
Professor of Laboratory Medicine, of Cell Biology and of Pathology; Assoc. Director, Yale Stem Cell Center; Assoc. Director, Transfusion Medicine Service; Medical Director, Clinical Cell Processing Laboratory; Medical Director, Advanced Cell Therapy Laboratory

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