11 Specimen Preparation Techniques for Cytogenetic Analysis by Fluorescence in situ Hybridization
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10. Repeat steps 7-8.
11. Incubate the tube on ice for 30 to 60 min.
12. Spinat 200 g in a cooled (4°C) centrifuge and repeat steps 7-9 at least 5
more times (keep tubes on ice).
13. Resuspend cells in a small volume ( e.g. 0.5 - 1 ml) flxative solution.
14. Drop the cells from a distance of about 10-50 cm on pretreated slides
(see above). Slides should be kept in a humidifled enviroment while the
cells are dropped. Check concentration of cells by microscopy. If the
concentration of cells is too high, add flxative solution, if it is too
low, spin again and resuspend in smaller volume.
15. Keep the slides in a humidifled chamber for another 5 min.
16. Air dry slides.
17. Dehydrate slides in a series of 70%, 90%, 100% ethanol for 5 min each.
18. Air dry again and keep slides at room temperature for one day.
19. Slides can either be used directly for in situ hybridization or stored at70°C for several month. Try to prevent water condensation when thawing the slides before FISH.
20. If no pepsin digestion step is included (see Subprotocol3) proceed with
fluorescence in situ hybridization protocol (see Chap. 12).
The penetration of probe molecules can be signiflcantly facilitated by mild
digestion with pepsin prior to FISH. This may be a critical step for obtaining
strong hybridization signals in clinical samples. Therefore, we describe a
brief method for pepsin digestion of chromosome spreads (Raap et al.
1992). The optimal amount of pepsin mayvary for different sources of cells.
Subprotocol 3
Pepsin Digestion of Blood lymphocytes
Materials
• Phosphate-buffered saline (PBS).
• 0.01 M HCl.
151
10. Repeat steps 7-8.
11. Incubate the tube on ice for 30 to 60 min.
12. Spinat 200 g in a cooled (4°C) centrifuge and repeat steps 7-9 at least 5
more times (keep tubes on ice).
13. Resuspend cells in a small volume ( e.g. 0.5 - 1 ml) flxative solution.
14. Drop the cells from a distance of about 10-50 cm on pretreated slides
(see above). Slides should be kept in a humidifled enviroment while the
cells are dropped. Check concentration of cells by microscopy. If the
concentration of cells is too high, add flxative solution, if it is too
low, spin again and resuspend in smaller volume.
15. Keep the slides in a humidifled chamber for another 5 min.
16. Air dry slides.
17. Dehydrate slides in a series of 70%, 90%, 100% ethanol for 5 min each.
18. Air dry again and keep slides at room temperature for one day.
19. Slides can either be used directly for in situ hybridization or stored at70°C for several month. Try to prevent water condensation when thawing the slides before FISH.
20. If no pepsin digestion step is included (see Subprotocol3) proceed with
fluorescence in situ hybridization protocol (see Chap. 12).
The penetration of probe molecules can be signiflcantly facilitated by mild
digestion with pepsin prior to FISH. This may be a critical step for obtaining
strong hybridization signals in clinical samples. Therefore, we describe a
brief method for pepsin digestion of chromosome spreads (Raap et al.
1992). The optimal amount of pepsin mayvary for different sources of cells.
Subprotocol 3
Pepsin Digestion of Blood lymphocytes
Materials
• Phosphate-buffered saline (PBS).
• 0.01 M HCl.