Protocol by Ross Francis
Modified by Sudhir Nayak
1) Pick adults and/or L4s to an unseeded NGM plate or directly into 1.5ml of PBS. Alternatively, wash worms off a seeded plate with 1.5 ml of PBS and allow to gravity settle for a few minutes in an eppendorf tube, then wash once with 1.5 ml of PBS. For RNA in situs, gonads from young adults generally survive better than those from old adults. Transfer as many worms as can be dissected in 5 min (<100, unless you are really good).
2) Suspend worms from one plate in 1-3 ml of PBS / 0.2 mM levamisole and transfer to a glass dish. The dish should have a relatively flat bottom and be large enough so that the worms are well spread out from one another. We use dishes (from Carolina Biological Supply) that look something like:
3) As paralysis sets in, begin cutting off heads at level of the pharynx. Place the head between two 25 guage syringe needles and decapitate by moving needles in a scissors motion (avoid needles with bent tips). For most animals, at least one gonad arm should extrude completely.
4) Remove excess liquid with a drawn-out pasteur pipette.
5) Fix the gonads:
with methanol for quick DAPI staining
with formaldehyde or methanol / formaldehyde for DAPI staining with improved morphology
with glutaraldehyde / formaldehyde for RNA in situ hybridization
PBS: Sambrook et al. (Molecular Cloning).
PBS / 0.2 mM levamisole (from 100 mM Levamisole stock).
levamisole (Sigma) stock: 100 mM in dH20 (store at -20).