Protocol by Ross Francis
Modified by Min-Ho Lee and Sudhir Nayak
A. Methanol
Methanol fixation is quick, easy, and gives satisfactory nuclear morphology after DAPI staining. However, methanol-fixed gonads break easily and are more difficult to mount than formaldehyde-fixed gonads. Fix dissected gonads in 3 ml of cold (-20) methanol for 5 min. Using a pasteur pipette, transfer to a glass conical glass tube, add 2ml PBT and spin 1 min in a clinical benchtop centrifuge at setting 2. Remove the supernatant and wash 2x in several ml PBT. Fixed gonads can be stored in methanol at -20 for a week or two before use.
B. Formaldehyde
Gives better nuclear morphology and less shrinkage than MeOH fixation. Fix in 2 ml of 3% Formaldehyde/0.1 M K2HPO4 (pH 7.2) for 10 min to 1 hr. (More recently we have found that a 10 min fix gives better detection for a number of antibodies.) After fixation, transfer to a 5 ml glass conical tube (the more narrow the bottom is, the better), add a few mls of PBTw and spin 1 min in clinical benchtop centrifuge at setting 3. Remove super, wash 1x in PBT, and then post-fix in 2 ml of 100% Methanol (from -20 C stock) for 5 min. Fill tube with PBT, spin, remove super ( I usually leave about 200 ul), and transfer the worms into a small (6 mm X 35 mm) glass culture tube (siliconized, Kimax brand) . Alternatively, fixed gonads can be stored in MeOH at -20C for a few days. Storing in methanol for longer periods is possible, but subsequent staining seems of a lower quality and nuclear morphology degrades.
If you want to retain some native GFP signal a 10 min in 3% formaldehyde/0.1 M K2HPO4 (pH 7.2) can be used for dissected gonad arms (no methanol post-fix). Arms will be more fragile than with the longer fix so care should taken when transfering.
C. Methanol / Formaldehyde
This fixation protocol is fast and gives better DAPI-nuclear morphology than methanol by itself. To make the fixative, mix 10 mls of 16% formaldehyde, 3.3 mls of 0.1 M K2HPO4 (pH7.2), and 40 ml methanol. This solution is stored at -20 and is used as described for methanol (but increase fixation time to 10 min).
D. Glutaraldehyde / Formaldehyde
Glutaraldehyde / formaldehyde fixation works well for germline RNA in situs: gonads remain intact through extensive protease treatments (40 ug/ml Proteinase K for 30 min) that would destroy gonads fixed in formaldehyde alone. This fixation has also been used with good results with some antibodies. Mix 10 ml of 16% formaldehyde, 0.53 ml of 25% glutaraldehyde (EM grade), and 43 ml of 0.1 M K2HPO4 (pH 7.2). Store the fixative in 5-10 ml aliquots at -20¡C. Fix gonads for 2 hr at rom temperature with a 5 min MeOH post-fix (as described above for formaldehyde).
Materials
PBS: Sambrook et al. (Molecular Cloning).
PBT: PBS containing 0.1% Tween 20.
3% formaldehyde / 0.1 M K2HPO4 (pH7.2): Prepare from sealed ampoules of 16% EM grade formaldehyde (EM Sciences). Freeze any excess. This solution can also be made from paraformaldehyde powder.
Methanol: 100% stock kept at -20C.
NOTE: For fixation of whole animals the same protocols can be used.