1. Centrifuge urine in a 50 mL centrifuge tube at 600 g for 10 minutes at room temperature (15—30ºC).
2. Remove the supernatant to within approximately 1—2 mL of the cell pellet, being careful not to disturb the pellet.
3. Resuspend the pellet in the remaining 1-2 mL of supernatant and transfer the contents to a 15 mL conical centrifuge tube. Rinse the 50mL tube with 10 mL of 1X PBS and transfer the contents to the 15 mL tube.
4. Centrifuge sample(s) at 600 g for 10 minutes at room temperature.
5. Remove the supernatant to within approximately 0.5 mL of the cell pellet.
6. Resuspend pellet in the remaining 0.5 mL of supernatant. Slowly add 1-5mL of fresh fixative (3:1, methanol:acetic acid), dropwise at first, with frequent agitation.
7. Let fixed specimens stand at —20ºC for a minimum of 30 minutes
8. Centrifuge sample(s) at 600 g for 5 minutes at room temperature. Carefully remove the supernatant.
9. Wash pellet by resuspending in 1-5 mL fixative.
10. Centrifuge sample(s) at 600 g for 5 minutes at room temperature. Repeat steps 8 & 9 twice.
11. After centrifugation of cell suspension in fixative:
    • If cell pellet is very small and hardly visible, CAREFULLY remove as much fixative as possible, leaving approximately 100 µL solution. 
    • If cell pellet is easily visible, remove as much fixative as possible and add 0.5-1 mL fresh fixative to the cell pellet.
12. Proceed immediately with the slide pretreatment protocol. Follow with FISH pretreatment kit. 

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