• The larval brain is the tissue of choice for the study of cell division during late development. Most mitotic mutants of Drosophila have been identified by screening squashed preparations of third-instar larval brains.
• We include here two protocols for immunofluorescent staining cells from the larval brain.

lmmunfluorescent staining by Squashed Preparations of larval Brains:

1. Dissect out larval brains in 0.7% NaCl.
2. Place the brain in a microcentrifuge tube containing 3.7% formaldehyde in phosphate-buffered saline (PBS), and incubate at room temperature for 1 hour.
3. Remove the formaldehyde, add 10% Fetal calf serum (FCS), 0.3%triton X-100 in PBS, and incubate for 1 hour at room temperature.
4. Place the 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 an 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.
   • Keep pressing for at least 10 seconds and release pressure gently.
   • Repeat, pressing on the other two comers.
7. Immerse the end of the slide carrying the coverslip in liquid N₂.
   • When the nitrogen ceases to boil, remove the slide and lever off the coverslip with the flick of a scalpel.
8. Dehydrate by successive immersion of the slides in 70% ethanol for 3 minutes, 100% ethanol for 3 minutes, and air dry before use.
9. Carry out immunostaining as described for in toto preparations.

lmmunostaining Whole-Mount Preparations of larval Brains:

1. Dissect out larval brains in 0.7% NaCl.
2. Incubate for 30 minutes in 3.7% formaldehyde followed by 60 minutes in 37% formaldehyde.
3. Transfer to 0.3% triton X-100, 10% FCS, in 0.7% NaCl, and keep there for about 20 minutes.
4. Incubate with the first antibody in 2.5 mg/ml RNAase, 0.3% triton X-100, 10% FCS, in 0.7% NaCl overnight
5. Wash in 0.3% triton X-100, 10% FCS, in 0.7% NaCl at least four times for 10 minutes.
6. Incubate with second antibody in 0.3% triton X-100, 10% FCS, in 0.7% NaCl at least for 2 hours.
7. Wash in 0.3% triton X-100, 10% FCS, in 0.7% NaCl at least four times for 10 minutes, and then in 10% FCS in 0.7% NaCl for a few minutes.
8. If required, incubate in propidium iodide 1in 0.7% NaCl for 5 minutes.
9. Transfer the brains into a drop of approximately 50 pl of mounting medium on a microscope slide, move them around with a pair of twizers until they are embedded, cover, and seal with nail varnish.

Immunofluorescent staining of Drosophila Larval Neuroblasts

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