34 Simultaneous Fluorescence Immunophenotyping and FISH (FICTION)
449
1. In a cooled Petri dish cut a piece of tissue (10-40 mg) from a tumor
block stored in liquid nitrogen or at -80
c e. Refreeze remaining tumor
material immediately!
2. Per 10 mg tumor tissue add 60-120 I.d 1x PBS (tissue-dependent).
3. Thaw tissue in 1xPBS for 15-30 min at RT.
4. Disaggregate tissue mechanically by cutting and scraping with a scalpel.
5. Collect 1x PBS solution containing the cells from the dish and transfer
it to a 1.5-ml Eppendorf tube.
6. Disaggregate cells in solution by pipetting several times through a
10-100 J!l pipette.
7. Count the number of cells in solution.
8. Transfer 3000 cells/slide into a new Eppendorf tube.
9. Add 200 J!l digestion solution. (Do not prewarm!)
10. Incubate at 37°C for 10 min.
11. Immediately transfer tube onto ice; cytocentrifuge the solution for
5 min at 800 rpm.
12. Air dry slides for 10 min.
13. Fix slides for 10 min in paraformaldehyde solution (1%).
14. Wash slides once in A. bidest.
15. Dehydrate in 70%, 85% and 100% ethanol for 1 min each.
16. Air dry slides.
Note: The remaining cell solution can be stored for several months.
Add 2.3 vol 100% ethanol and freeze at -20°e. For preparing slides
from this stored cell solution, transfer required amount of cells
into an Eppendorf tube, pellet cells by short spinning in a dish centrifuge, discard supernatant, add 40 J!11 xPBS per 1x 10
5
cells and incubate on ice for at least 15 min. Proceed with step 9.
449
1. In a cooled Petri dish cut a piece of tissue (10-40 mg) from a tumor
block stored in liquid nitrogen or at -80
c e. Refreeze remaining tumor
material immediately!
2. Per 10 mg tumor tissue add 60-120 I.d 1x PBS (tissue-dependent).
3. Thaw tissue in 1xPBS for 15-30 min at RT.
4. Disaggregate tissue mechanically by cutting and scraping with a scalpel.
5. Collect 1x PBS solution containing the cells from the dish and transfer
it to a 1.5-ml Eppendorf tube.
6. Disaggregate cells in solution by pipetting several times through a
10-100 J!l pipette.
7. Count the number of cells in solution.
8. Transfer 3000 cells/slide into a new Eppendorf tube.
9. Add 200 J!l digestion solution. (Do not prewarm!)
10. Incubate at 37°C for 10 min.
11. Immediately transfer tube onto ice; cytocentrifuge the solution for
5 min at 800 rpm.
12. Air dry slides for 10 min.
13. Fix slides for 10 min in paraformaldehyde solution (1%).
14. Wash slides once in A. bidest.
15. Dehydrate in 70%, 85% and 100% ethanol for 1 min each.
16. Air dry slides.
Note: The remaining cell solution can be stored for several months.
Add 2.3 vol 100% ethanol and freeze at -20°e. For preparing slides
from this stored cell solution, transfer required amount of cells
into an Eppendorf tube, pellet cells by short spinning in a dish centrifuge, discard supernatant, add 40 J!11 xPBS per 1x 10
5
cells and incubate on ice for at least 15 min. Proceed with step 9.