Fig. 5: Crystals preferentially disrupt amyloid dye-staining material of the pharyngeal cuticle.

A–F Images of the anterior-most end of the worm. The imaging channel is indicated in each panel in the lower left as is the wactive treatment (left-hand side of micrograph series). DIC, differential interference contrast imaging; bire, birefringence imaging; CFW, calcofluor white; ThS, thioflavin S; EY, eosin Y. Merge 1 is the merge of the two dye signals. Merge 2 is the merge of the signals from the amyloid-binding dye and birefringence. Blue arrows indicate overlapping signals of the birefringent crystals and the amyloid-binding dye and highlights the staining of abnormally displaced material; orange arrows show a lack of amyloid-binding dye signal associated with birefringence signal. Note that in (A–C), the panels show the overlap between birefringence signal and CWF and CR staining, while in (D–F), the panels show the overlap between birefringence signal and EY and ThS staining. Similar results were obtained with wact-190 over 6+ trials, and with wact-390 over 2 trials. G Control for the ability of dyes to bind wactive crystals. Precipitate of wact-190 or wact-390 was formed in solution (or DMSO solvent control) and was subsequently stained with the dye indicated on the right of each set. The dyes were used at the same concentration as the in vivo staining experiments as described in the methods. Each condition was imaged using DIC, birefringence (bire.) or the appropriate fluorescent (floures.) channel, as indicated on the left of each panel. The dyes were not rinsed from the slide, which accounts for the non-specific signal in the EY fluorescent channel. The scale in the upper left micrograph applies to all images in (G). H, I Controls showing that the crystals themselves fail to fluoresce in the different channels used for imaging; D, differential interference contrast; B, birefringence; D, DAPI; C, CFP; G, GFP; Y, YFP; R, RFP channels. The scale bar in (H) also applies to (I). 60 μM of wact-190 and wact-390 was used in all experiments relevant to panels (A–I). Similar results were obtained for (G–I) over 3 trials.