• In situ hybridization to diploid cells serves in mapping and characterizing the behaviour of heterochromatic sequences of Drosophila, which are both under represented and aggregated in polytene cells.
•  Two protocols are included for in situ hybridization to diploid cells from squashed and whole-mounted third-instar larval brains.
  • For genome mapping, squashed preparations are preferred as they are easy to make and provide the highest possible resolution.
  • The procedure can be employed either in untreated brains or in brains where the number of mitotic chromosomes has been artificially increased by incubation with colchicine.
  • Whole mounted protocol should be carried out when more functional studies are to be performed .
  • Whole-mounted tissues conserve the original three-dimensional arrangement of subcellular organelles and are devoid of the artefacts produced by squashing.
  • These are necessary requirements needed to study problems like chromosome pairing, chromosomal domains in interphase nuclei, attachment sites, and many others.

Fluorescent in-situ Hybridization to Squashed Diploid Cells:

1. Obtaining brain of larvae:
   • With a pair of tweezers, brains are obtained from third-instar larvae by pulling from the mouth parts while holding the larvae by their middle.
   • The brain (ventral ganglion plus optic lobes) usually comes out together with the salivary glands and other internal tissues.
   • Carefully remove other tissues as brain preparations devoid of other tissues are essential to obtain good squashes.
2. Dissect out larval brains in 0.7% NaCl and incubate in 0.5 kg/ml colchicine in 0.7% NaCl in a dark, humid chamber for 2 hours.
3. Apply hypotonic shock by washing in 0.5% trisodium citrate for 10 minutes.
4. Place the dissected brains on a microscope slide, add a drop of 45% acetic acid, and leave for 30 seconds.
5. Remove the liquid using tissue paper, add a drop of 60% acetic acid, and cover with a 18 X 18 mm² coverslip.
   • Do not squash yet, but wait for 3 minutes.
6. Squash between two sheets of blotting paper by pressing with two fingers on opposite comers.
7. Keep pressing for at least 10 seconds and release pressure gently. Repeat, pressing on the other two comers.
8. Immerse the end of the slide carrying the coverslip in liquid N₂.
   • When the nitrogen ceases to boil, remove the slide and level off the coverslip with the flick of a scalpel.
9. Dehydrate by successive immersion of the slide in 70% ethanol for 3 minutes, 100 %ethanol for 3 minutes, and air dry before use.
10. Bake the slide at 58°C for 1 hour in a dry oven.
11. Immerse slides in H₂O gently to remove the coverslips.
12.  Denature the chromosomal DNA by boiling for 3 minutes.
    • The slides should be kept in hot (>80°C) water until the probe is applied.
13. Apply 10 µl denatured probe and carry out the hybridization at 58°C in a humid chamber overnight
14.  After hybridization, carry out the washing and Fluorescein isothiocyanate (FITC) staining.

Fluorescent in Situ Hybridization to Whole-Mounted Diploid Tissues

1. Dissect brains in saline.
2. Fix for 10 minutes in formaldehyde 3.7% in saline.
3. Transfer to an Eppendorf tube and add about 500µl of 37% formaldehyde and 200 µl of n-heptane.
4. Incubate for 20 minutes in spinning wheel at room temperature.
5. Remove all the heptane and as much formaldehyde as possible, add 500 µl of 1% triton X-100 in PBS, and incubate for 20 minutes.
6. Remove supernatant and wash once in phosphate-buffered saline (PBS)
7. Add 50 of probe and cover with 50 parafilm oil.
8. Denature in boiling water bath for 10 minutes and incubate overnight at 58°C.
9. Add 4X saline sodium citrate (SSC)  under the oil and remove as much oil as possible.
10. Wash for 10 minutes in 4X SSC.
11. Wash for 10 minutes 4X SSC + 0.1% triton X-100.
12. Wash for 10 minutes in 4X SSC.
13. Incubate in 150 FITC-avidin in 4X SSC for 30 minutes at room temperature and repeat washes as in steps 10 to 12.
14. Incubate in 1 propidium iodide in 4 X SSC for 10 minutes.
15. Wash for 10 minutes in 4X SSC.
16. Mount in glycerol-propyl gallate.

Fluorescent in-situ Hybridization to Diploid Cells

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