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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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