Method prepared by Grace Panganiban:

X-gal staining imaginal discs

Solutions needed:
1x PBS pH 7.0 (make up ~100 mls fresh from 10x stock)
25% glutaraldehyde (store at -20oC and handle with care, this is nasty stuff!)
30% glycerol
8% X-gal in DMSO (store in 25 ul aliquots at -20oC; each aliquot will make 1 ml of staining solution)
Staining solution:
10 mM NaH2PO4.H2O/Na2HPO4.2H2O (pH 7.2)
150 mM NaCl
1 mM MgCl2
3.1 mM K4[FeIII(CN)6]
3.1 mM K3[FeII(CN)6]
0.3% Triton X-100

Tools needed
9-well dissecting dish
24 or 96 well microtiter dish
2 prs of watchmaker's forceps
pasteur pipettes

1) Prewarm staining solution (~300 ul for each sample) to 37oC.

2) Make up fixative (~500 ul for each sample) using 30 ul of 25% glutaraldehyde for each ml of PBS.

3) Fill the wells of the dissecting dish with PBS.

4) Fill one well per genotype of the microtiter dish with PBS.

5) Place several 3rd instar larvae in one of the wells of the dissecting dish. The larvae will float in 30% glycerol. If necessary, you can flood the vials with glycerol to get them out of the food. After rinsing them with PBS, transfer the larvae to a clean well containing PBS for dissection.

6) Hold the head of the larva just behind the mouthparts with one pair of forceps. Place the other pair of forceps about halfway down the body. Pull the forceps apart to rip the larva in half. While continuing to hold the head with the first pair of forceps, insert one of the tines of the second pair of forceps into the open end of the larva, grab the cuticle, and pull again to expose the brain, the salivary glands and the disc complexes. After cleaning off as much of the fat (opaque white tissue) as possible, transfer the disc complexes (which may still be attached to the brain or the head skeleton) to the microtiter dish. Put ~5-10 complexes in each well.

7) Once you have dissected the discs from each genotype to be stained (but within 2 hrs), remove the PBS from each microtiter well, and replace it with fixative.

8) After 15-20 minutes, remove the fix, and rinse the discs 1x with PBS.

9) Add 1 ml of staining solution to each tube of X-gal.

10) Remove the PBS from the discs, and add ~300 ul of staining solution to which the X-gal has been added.

11) Allow the discs to develop at 37oC for several hours-several days.

12) Stop the reaction by rinsing discs with PBS.

13) Store the discs at 4oC in PBS (or PBS+10% glycerol).

14) Dissect discs in a droplet of PBS (or PBS+10% glycerol) on microscope slides. Discard all excess tissue that could prevent the coverslip from flattening the discs. Cover the discs with a coverslip; blot away the excess mounting medium; and seal the coverslip with nailpolish.

15) Examine/photograph your samples within a few days. They do not store well.


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