Strings and webs stain with ThT and have a beaded structure: LM/SEM. (A) Live growth-arrested ScGT1 were stained at 8°C with 8B4 followed by RRX secondary Fab. PM sheets were then transferred to glass slides, fixed, stained with ThT, and examined for the fluorescence shift indicative of amyloids. ThT staining (b, inset, green) colocalized with 8B4-labeled strings (a); merge shown in c. Bar: (a, inset) 0.5 µm. (B–D) Quasi periodic immunostaining of strings/webs. Fixed SMB (B) and fixed/FA-treated ScGT1 (C and D) cells were stained with the indicated Abs. Both N- and core Abs had a punctuated distribution hinting at a “beaded chain.” In some cases, individual foci were especially prominent with one Ab but not with the other (C, b, arrows), suggesting local differences in the ratio of FL/trimmed PrP^Sc. (B) Several z-planes 300 nm apart (such as the one in the left panel) were acquired. 3D reconstructions show quasi-periodic immunostaining (right, arrow) of strings crossing the focal plane (arrowheads). The distance between consecutive peaks is 300–600 nm. (D) In many instances, strings organized in webs of various complexities. Some strings appeared to serve as organizing “stems” (arrow), which sprouted ramifications (D, b, arrowheads). (E) Correlative LM/SEM. Live ScGT1 (a–c) growing on ITO-coated coverglass slides were treated with PIPLC and then stained with 8B4 followed by fluorescent and 18-nm immunogold labels before fixation (Materials and methods). (d) ScGT1-PPS (not treated with PIPLC) were labeled as above, except that a clustering Ab was used to produce PrP^C patches on the cell surface. Cells were fixed, visualized by immunofluorescence (a, inset), postfixed, and further processed for SEM-BSe analysis without metal coating. (a) Strings seen by immunofluorescence (inset) correlated well with thin lines of immunogold in SEM-BSe (arrows and circles). Additional examples of strings are shown in b and c. In contrast, patched PrP^C appeared as unorganized clumps of immunogold (d).