Larval Brain Squashes
Larval Brain Squashes
Making squashes
1.Pick 3rd instar larvae (4-5 day old in bottles). If applicable, use GFP scope if applicable to find appropriate genotype
2.Dissect larval brains in glass dish in 20% newborn calf serum (optional) and 80% of 0.7% Saline (1.4 g NaCl in 200 mL ddH20).
3.Move brains to a puddle of serum/saline on glass slide. Dissect other tissues away from brains. Put each pair of brains in a small Petri dish containing 1.5 mL of serum/saline [Option: use a six-well tissue culture plate.]
4.Add 6 μl of 0.025 M stock of colchicine for a final concentration of 0.1 mM.
[12 μl if using TC plate.]
5.Incubate for 1.5 hours on benchtop. 30 min. gives minimal mitotic spreads; increasing incubation time increases number of mitotic cells/spread.
6.Remove solution and add 1.5 mL of 0.5% citrate to induce swelling. Put on bench top and soak for 8 minutes. More than this often causes the tissue to “dissolve” and the sister chromatids to separate; too little time doesn’t allow for the chromosomes to “spread” easily for viewing.
7.Fix by using foreceps and dipping individual brains in a solution of acetic acid, methanol, and ddH20 (5.5:5.5:1) for 10-20 seconds.
~1 mL = 450 μl acetic acid, 450 μl methanol, 82 μlddH20
8.Transfer each to a corner of a large 22x40 mm coverslip. Put one pair in each corner, for a total of 4 brains on each large coverslip. Be sure to mark which side has the brain squashes
9.Put 2μlof 45% acetic acid on each brain. Let sit for ~2 min.
10.Put small 22 x 22 mm siliconized microscope coverslip on top. Squash with thumbs against bench top. Put a glass slide under the two coverslips for support and to avoid shattering the coverslips.
11.Lay the entire slide with “coverslip” sandwich on block of dry ice to freeze for 10-15 minutes. Small coverslip should easily pop off of the larger coverslip, with brain squashes stuck to the larger coverslip.
12.Soak large coverslip with squashes in 95% EtOH at -20oC to dehydrate. Air dry coverslip and store at 4oC if desired.
Viewing squashes
1.Rehydrate in 2X SSC for five minutes. Use the standing jar made for slides and soak in ~30 mL.
2.Stain with 0.1 ug/mL DAPI for five minutes:
3 μl working stock of DAPI in 30 mL of 2X SSC
3.Rinse ~5 seconds in fresh 2X SSC. Air dry in vertical position.
4.Mount large coverslip on a clean glass slide with Fluoromount G (one small drop per slide) and seal with clear nail polish. It is important to reduce the amount of volume between the slide and the large coverslip.
5.Slides can be stored at 4oC in dark or viewed immediately. Use 40X to find squashes, which will look like blue dust under epifluorescence. Use 100X oil immersion lens to look at individual nuclei.