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Page designed
and maintained by Octavian Henegariu (Email:
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WARNING:
The information provided in these pages is copyrighted and is intended
for individual use only. No parts of this work (text, tables or
pictures) may be commercialized, published or otherwise reproduced
without the written consent of the author. |
FISH
Guide and Troubleshooting
(fluorescent DNA probes)
a.
Slide preparation guide
Topics: #1
| #2
| #3
| #4
| #5
| #6
| #7
| #8
| #9
||| Figures: Fig1
| Fig2
| Fig3
| Fig4
| Fig5
| Fig6
|
| 1.
Cytogenetic
techniques introduction (short description of the cytogenetic protocols) |
6.
Slide
preparation (step-by-step protocol details) |
| 2.
Material
and Methods (from slide preparation to FISH detection) |
7.
When
do chromosomes spread? (description of what is happening on slides) |
| 3.
Cell
suspensions (preparation, handling, storage tips) |
8.
G-banding (slide storage and tips for banding) |
|
4.
Cell
fixatives |
9.
Selected
references (list of some literature titles used) |
| 5.
Slide
preparation factors (before, during and after dropping the cells) |
b.
FISH guide
TOPICS:
| 1
| 2
| 3
| 4
| 5
| 6
| 7
| 8
| 9
| 10
| 11
| 12
| 13
| 14
| 15
| 16
| 17
| 18
| 19
| 20
| 21
| 22
| 23
FIGURES: | 1
| 2
|
3 |
4 |
5 |
6 |
7
| 8
| 9
| 10
| 11
| TABLES:
| 1
| 2
| 3
| 4
| 5
| 6
| 7
| 1. Materials and Methods; Table 1,2,3 (introduction, summary of procedures) | 13. The "background theory"; Table 7, Fig 5,8,9 (why it happens, how to prevent) |
| 2. Chemical aging of slides; Fig 1,2,3,4 (hot ethanol pretreatment protocol) | 14. Cell quality in FISH; Fig 2 (comparison of quality of cells) |
| 3. Protease digestion; Fig 1,2 (pepsin or trypsin pretreatment protocols) | 15. Metaphase, interphase mapping; Fig 9 (simple procedures used in our laboratory) |
| 4. Slide denaturing (description of alternative protocols) | 16. FISH with short DNA probes; Fig 9 (use of plasmids, PCR products) |
| 5. Hybridization buffers; Table 6, Fig 7 (composition and use) | 17. FISH with complex probes; Fig 10, 11 (includes CGH, M-FISH, µdissection) |
| 6. Repetitive sequences blocking; Table 5, Fig 8 (use of Cot1 DNA, salmon sperm DNA) | 18. FISH with commercial probes; Fig 6, 10 (tips to decrease costs, do more tests) |
| 7. Posthyb wash, DAPI (wash conditions, antifade, DAPI banding) | 19. FISH on DNA fibers; Fig 10 (experimental tips, discussion) |
| 8. Detection: antibody, tyramide; Table 7, Fig 9, 10 (fluorescent antibodies, TSA comparison) | 20. FISH on paraffin sections; Fig 11 (includes DNA and RNA hybridization) |
| 9. DNA labeling (nick translation, PCR); Table 4, 5, Fig 5 (comparison of labeling methods) | 21.
FISH
and immunofluorescence (simultaneous protocol description) |
| 10. Labeled DNA purification (discussion of alternative procedures) | 22.
Multicolor
immunofluorescence (short procedure) |
| 11. Inter-Alu PCR, Alu banding; Fig 9, 10 (use of Alu repeats in human cytogenetics) | 23.
Selected
references (list of some literature titles used) |
| 12.
Multicolor
FISH; Fig 9, 10 (combinatorial labeling, triple color, multiple) |
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