Worm Breeder's Gazette 11(3): 79
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
We would like to share some of our immunofluorescence techniques with the worm world. We have developed a hydrogen peroxide treatment that permeablizes worms faster, more reproducibly, and much less expensively than collagenase, and a technique that greatly reduces photobleaching of fluorescein-labelled reagents. H202 permeabilization The idea behind this procedure is to reduce the disulfide linkages that help hold the cuticle together, and then quickly oxidize the -SH groups to -S03 before the disulfides can reform. Cysteines and methionines in other proteins will be oxidized as well, possibly affecting some epitopes. Met but not cys can be restored by a second DTT treatment after the oxidation step. 1. Fix animals. We use formaldehyde, as described in Ruvkun and Guisto, Nature 338, 313-319, 1989; other fixation protocols will probably work. Overfixation seems to give a high background with this technique. 2. Reduce disulfides to -SH: Wash worms once (in a microfuge tube) with Tris Triton buffer, then incubate in Tris Triton buffer + 1% ME, 37 C 1 hr with agitation. After this point the worms are fragile and shouldn't be spun hard. Wash worms once in 1x BO3 buffer, then incubate in BO3 buffer + 10mM DTT, 15 min 37 C with agitation. 3. Oxidize -SH groups to -S03: Wash worms once in BO3 buffer, then incubate in BO3 buffer + 1% H2O2, 1 hr at room temperature. Agitate gently but keep tubes upright because the cap may pop open from O2 pressure. Wash once with BO3 buffer and once for at least 15 min with antibody buffer B. Store worms in antibody buffer. 4. To check that worms are permeable to macromolecules, incubate an aliquot in 10 g/ml RNase A 1 hr 37 C, then stain RNased and unRNased animals with 1% Toluidine blue and rinse several times in buffer B. Compare the two samples at 50x with a dissecting scope. Open animals will have very little staining of internal tissues. 5. Do antibody incubations in buffer A and washes in buffer B. Permanent Springtime mounting procedure 1. Make a pad of Permanent Springtime agarose on a slide. 2. Put a 3-5 l drop of NPG solution on the pad, add an equal volume of stained worms, and mix with a pipet tip. 3. Cover with a cover slip and examine using epifluorescence. After an hour or two the DAPI stain starts to yellow and 'bleed through' to the FITC channel, so don't mount too many samples at once. [See Figure 1]